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Magnetic fluids or ferrofluids, are comprised of magnetic nanoparticles stabilized by coating surfactants and dispersed in various media, most notably hydrocarbons, esters or water [1 - 5]. The stability of magnetic fluid depends upon a balance between repulsive and attractive interactions among nanoparticles . Besides the thermal motion, the steric and electrostatic repulsive interactions are against van der waals and dipolar attractive interactions . The magnetic properties of magnetic fluids depend strongly on the size of the particles and the concentration of the magnetic material in the dispersion . In the presence of a magnetic field, the magnetic moment of the particles will try to align with the magnetic field direction leading to a macroscopic magnetization of the fluid . When the external field is removed, the particles quickly randomize the directions of their magnetic moment and the fluid loses its magnetism . The study and application of magnetic fluids were invented in the mid-1960s, involving the multidisciplinary sciences such as chemistry, fluid mechanics, and magnetism . With modern advances in understanding nanoscale systems, iron oxide fluids in hydrophobic media are now used industrially for rotating shaft seals, loudspeaker coils, and in various magnetically promoted separations . However, the magnetic properties of iron oxide - based fluids are not sufficient for a number of purposes . A variety of techniques have been developed to fabricate magnetic fluid using metal particles such as spark erosion and vacuum evaporation . One of the major difficulties encountered is that the magnetization of the metal particles decays with time, due to the lack of oxidization resistance in the ambient environment . According to nakatani et al ., the iron - nitride compounds are ferromagnetic with higher magnetization than iron oxide (fe3o4) and are chemically stable . They are metallic with a close packed lattice of iron atoms whose interstices are occupied by nitrogen atoms . It is considered that there exists covalent bonding between the iron atoms and the nitrogen atoms . Thus, fine particles of ferromagnetic iron - nitrides have a potential application to magnetic fluids that need a high magnetization and stability against oxidation . In this article, single - phase -fe3n - based magnetic fluid and nanometer powders were analyzed . In the magnetic fluids, the assays performed for the preparation, stability, and evaluation of the magnetic fluids are discussed in the following sections . Iron - nitride - based magnetic fluid was synthesized according to the method reported in ref . . The carrier liquid was composed of 80-ml -olefinic hydrocarbon synthetic oil (pao4 oil with low volatility and low viscosity) and a certain amount of succinicimide (surfactant) (see table 1). The only difference is that the carrier liquid used in powder preparation was synthetic oil without any surfactant . It is convenient to collect the dried particles when they are not coated by surfactant using a magnet . The gas flux ratio was fixed at ar1:ar2:nh3 = 7:2:2 where ar1 and ar2 represent argon gas coming from two gas pipes according to the ref . . The argon gas ar1 was used to carry the iron carbonyl which was heated to gasify at 37.5 c . The argon gas ar2 was used to control the concentration of iron carbonyl vapor and ammonia gas in the mixed gas . The surfactant content in five magnetic samples (fn001fn005) xpert pro (panalytical) x - ray diffractometer was used to analyze the phase composition of magnetic particles . The diffraction was performed with cok1(= 1.7889) and the ray was filtered by the graphite . The experimental parameters used were: 40 ma, 35 kv, continuous scan, scan speed 2/min . Particle sizes of iron - nitride coated with surfactant were measured with a 2,100 fx transmission electron microscope (tem) operated at 200 kev . Tem samples were prepared by dispersing the particles in alcohol using ultrasonic excitation, and then transferring the nanoparticles on the carbon films supported by copper grids . The density of the magnetic fluid was measured using a picnometer at 20 1 c . The sedimentation stability of the magnetic fluid was evaluated by changing the ratio of the synthetic oil (pao4) and surfactant . After obtaining stable magnetic fluid, the magnetic properties of the magnetic powders and the magnetic fluids were measured immediately after preparation using a ldj9500 vibrating sample magnetometer (vsm). Samples were contained in a small glass cup with internal dimensions of 2 2 2 mm, which were sealed by gluing a small cover glass over the open end . Then the glass cup was put in the magnetic uniform area of the pole . The hysteresis curve was recorded per 1.5 s. all measurements were performed at room temperature . The relationship between the calculated / measured saturation magnetization and particle content is also discussed ., we can see that the sites and intensity of the diffraction peaks are consistent with the standard pattern for -fe3n (jcpd no.1 - 1236) but it is obvious that every peak is broadened . According to the scherrer relationship, dhkl = 0.9/bcos (bis the half width of xrd diffraction lines, = 1.7889, is the half diffraction angle of 2), particle size was calculated . In this equation the average particle sizedhklwas determined by taking an average particle size ofd002,d101,d102, and the average particle size of 18.9 2 nm is obtained . X - ray diffraction pattern of -fe3n magnetic particle (cok1) figure 2a shows tem images of -fe3n particles covered with surfactant . It can be seen that the -fe3n particles synthesized in our experiment are spherical and have very narrow size distributions . Every particle is covered completely by the surfactant and dispersed in the carrier liquid homogeneously . The average particle size is about 21 1 nm which approximate the sizes calculated by the scherrer formula . It is known that uncovered nanoparticles are easy to agglomerate for the high surface energy . 2b are not covered with a surfactant and they formed an agglomerate structure which caused the image in fig . The particles appear as clusters with average particle size of 23 2 nm which are larger than the particles coated with surfactant . Since the carrier liquid is only 100 ml -olefinic hydrocarbon synthetic oil, the -fe3n particles cannot be covered by surfactant and the particle s collisions lead to the formation of larger magnetic particles . As -fe3n nanoparticles are ferromagnetic, one possible explanation of the difference between calculated value from xrd and observed particle size is due to particle aggregation under the influence of the electromagnetic field in the tem while x - ray line broadening analysis discloses the size of the primary particles . Compared with the two tem images, we can see that the -fe3n particles could be dispersed as single particles in -olefinic hydrocarbon synthetic oil with the aid of succinicimide as surfactant . (a) coated with surfactant, (b) without any surfactant the stability is one of the most important properties for magnetic fluid and it will strongly affect the service life ., five -fe3n magnetic fluid samples were prepared and all the reaction conditions were the same except the content of the surfactant in the carrier liquid given in table 1 . After preparation, the initial density of the five magnetic fluid (ini) samples was measured . Resting all the magnetic fluid samples above in a tube for a period of time, we then move 0.9 volume of the fluid to the top of the tube . Bot represents the density of the fluid in the bottom one tenth of the tube after resting for a period of time . Then the stability of the fluids was roughly estimated from the percentage of particles suspended and it was calculated as eq . 1:(1) where s% expresses the suspension percentage of the magnetic particles in fluid . After resting for 30 days, bot of the five magnetic fluids with different surfactant were measured . The particle suspension percentage (s%) of the five magnetic fluid samples was obtained using eq . 1and the relationships between s% and surfactant content in the carrier liquid are given in fig . 3 we can see that suspension percentage (s%) increases with the increase of surfactant content . When the volume fraction of the surfactant in carrier liquid reaches 20%, the s% calculated by eq . It slightly decreases to 95% when the volume fraction of the surfactant reaches 23.8% and this may be caused by measurement error . According to eq . 1, the suspension percentage of samples fn001fn003 is lower and is due to the higher value of bot . This means that the particles cannot be coated completely by the surfactant due to the deficiency of surfactant content and part of them have settled to the bottom of the tube . When the volume ratio of surfactant was in the range of 2023.8% (samples fn004 and fn005), the suspension percentage is higher . This means that the magnetic particles can be coated completely by the surfactant and dispersed in the carrier liquid homogeneously (see fig . 1and surfactant amounts the stability of samples fn004 and fn005 versus time is given in fig . It can be seen that suspension percentage of the two samples decreases quickly in the first 4 months . The changes ofs% for samples fn004 and fn005 are 3.5% and 2.0%, respectively . Both of the suspension percentages of samples fn004 and fn005 are kept above 92% calculated using eq . The relationship between suspension percentage and resting time in this part, five stable -fe3n magnetic fluids with different particle content were prepared using 80-ml synthetic oil (pao4) and 20-ml surfactant . Figure 5 exhibits the room temperature hysteresis loops of pure -fe3n nanoparticles samples under the field up to 10 koe . Though the 10-koe field may not be high enough to saturate the sample, the powder sample approaches to saturation and the value is about 83.5 emu / g, which can be considered as the saturation magnetization (s). In fig . 5 we can see that there is a small amount of hysteresis loss in the curve and this may be caused by the size of the particles . For the particles with no surfactant during preparation, the size of part of the particles in the powder sample is larger than the single domain dimension which will show some ferromagnetic loss . The coercivity value (hc) of the particles was measured to be 129 oe . According to viota et al ., the volume fraction () of a solid phase in magnetic fluid can be obtained by measuring the magnetic fluid density (). 2:(2) where f (0.84 g / cm) is the density of the liquid phase and s(6.88 g / cm) is the density of solid phase . Then the volume fraction () of the solid phase in magnetic fluid is(3) hysteresis loops at room temperature of the -fe3n nanoparticles under the field up to 10 koe (s = 83.5 emu / g) if the interaction between magnetic particles in a certain magnetic fluid is ignored, the saturation magnetization of a magnetic fluid should be considered as the sum of the magnetization of all the dispersed magnetic particles . Taking the density () as a variable, the value of the saturation magnetization (emu / g) for magnetic fluids can be expressed as:(4) where sand are the magnetization of the dispersed particles and magnetic fluid, respectively . Figure 6 gives the relations between the calculated volume fraction () of particles and the density of the magnetic fluid samples using eq . It can be noticed that the density of the fluid increases linearly with increasing of iron - nitride magnetic particles in the fluid . The measured magnetization and the calculated values using eq . 4 for the different volume fractions of the -fe3n - based magnetic fluids it can be seen that both the calculated and measured magnetizations increase with increasing of the particles in the fluid . At the low concentrations, the measured values are close to the calculated one . With the increasing volume fraction of the -fe3n particles, the measured the phenomenon may be caused by two reasons: (1) particle volume fraction, (2) particle size . In magnetic fluid, in addition to the hydrodynamic interaction, there exists the dipolar magnetic dipolar interactions between magnetic particles are neglected and the magnetic particles in the fluid feel only the external magnetic field fitting the hypothesis of eq . Dipolar interaction of particles cannot be ignored and this kind of interaction may affect particles relative motion when magnetized . The interaction between magnetic dipoles increases with the increase of particle concentration and the magnetic moments of part of the particles that are not fully aligned with the magnetic field . The magnetic fluid samples are not yet saturated during the measurement . Due to the wide particle size distribution, thus, the super paramagnetic fraction present in the samples might reduce the magnetization since the magnetic measurements were performed at room temperature . The effect of particle volume fraction () on density and magnetization of magnetic fluids, we can see that the sites and intensity of the diffraction peaks are consistent with the standard pattern for -fe3n (jcpd no.1 - 1236) but it is obvious that every peak is broadened . According to the scherrer relationship, dhkl = 0.9/bcos (bis the half width of xrd diffraction lines, = 1.7889, is the half diffraction angle of 2), particle size was calculated . In this equation the average particle sizedhklwas determined by taking an average particle size ofd002,d101,d102, and the average particle size of 18.9 2 nm is obtained . X - ray diffraction pattern of -fe3n magnetic particle (cok1) figure 2a shows tem images of -fe3n particles covered with surfactant . It can be seen that the -fe3n particles synthesized in our experiment are spherical and have very narrow size distributions . Every particle is covered completely by the surfactant and dispersed in the carrier liquid homogeneously . The average particle size is about 21 1 nm which approximate the sizes calculated by the scherrer formula . It is known that uncovered nanoparticles are easy to agglomerate for the high surface energy . 2b are not covered with a surfactant and they formed an agglomerate structure which caused the image in fig . The particles appear as clusters with average particle size of 23 2 nm which are larger than the particles coated with surfactant . Since the carrier liquid is only 100 ml -olefinic hydrocarbon synthetic oil, the -fe3n particles cannot be covered by surfactant and the particle s collisions lead to the formation of larger magnetic particles . As -fe3n nanoparticles are ferromagnetic, one possible explanation of the difference between calculated value from xrd and observed particle size is due to particle aggregation under the influence of the electromagnetic field in the tem while x - ray line broadening analysis discloses the size of the primary particles . Compared with the two tem images, we can see that the -fe3n particles could be dispersed as single particles in -olefinic hydrocarbon synthetic oil with the aid of succinicimide as surfactant . The stability is one of the most important properties for magnetic fluid and it will strongly affect the service life . The stability of -fe3n - based magnetic fluid was investigated . In this part, five -fe3n magnetic fluid samples were prepared and all the reaction conditions were the same except the content of the surfactant in the carrier liquid given in table 1 . After preparation, the initial density of the five magnetic fluid (ini) samples was measured . Resting all the magnetic fluid samples above in a tube for a period of time, we then move 0.9 volume of the fluid to the top of the tube . Bot represents the density of the fluid in the bottom one tenth of the tube after resting for a period of time . Then the stability of the fluids was roughly estimated from the percentage of particles suspended and it was calculated as eq . 1:(1) where s% expresses the suspension percentage of the magnetic particles in fluid . After resting for 30 days, bot of the five magnetic fluids with different surfactant were measured . The particle suspension percentage (s%) of the five magnetic fluid samples was obtained using eq . 1and the relationships between s% and surfactant content in the carrier liquid are given in fig . 3 we can see that suspension percentage (s%) increases with the increase of surfactant content . When the volume fraction of the surfactant in carrier liquid reaches 20%, the s% calculated by eq . It slightly decreases to 95% when the volume fraction of the surfactant reaches 23.8% and this may be caused by measurement error . According to eq . 1, the suspension percentage of samples fn001fn003 is lower and is due to the higher value of bot . This means that the particles cannot be coated completely by the surfactant due to the deficiency of surfactant content and part of them have settled to the bottom of the tube . When the volume ratio of surfactant was in the range of 2023.8% (samples fn004 and fn005), the suspension percentage is higher . This means that the magnetic particles can be coated completely by the surfactant and dispersed in the carrier liquid homogeneously (see fig . 1and surfactant amounts the stability of samples fn004 and fn005 versus time is given in fig . It can be seen that suspension percentage of the two samples decreases quickly in the first 4 months . The changes ofs% for samples fn004 and fn005 are 3.5% and 2.0%, respectively . Both of the suspension percentages of samples fn004 and fn005 are kept above 92% calculated using eq . 1after 6 months and they can be considered stable . The relationship between suspension percentage and resting time in this part, five stable -fe3n magnetic fluids with different particle content were prepared using 80-ml synthetic oil (pao4) and 20-ml surfactant . Figure 5 exhibits the room temperature hysteresis loops of pure -fe3n nanoparticles samples under the field up to 10 koe . Though the 10-koe field may not be high enough to saturate the sample, the powder sample approaches to saturation and the value is about 83.5 emu / g, which can be considered as the saturation magnetization (s). In fig . 5 we can see that there is a small amount of hysteresis loss in the curve and this may be caused by the size of the particles . For the particles with no surfactant during preparation, the size of part of the particles in the powder sample is larger than the single domain dimension which will show some ferromagnetic loss . The coercivity value (hc) of the particles was measured to be 129 oe . According to viota et al ., the volume fraction () of a solid phase in magnetic fluid can be obtained by measuring the magnetic fluid density (). 2:(2) where f (0.84 g / cm) is the density of the liquid phase and s(6.88 g / cm) is the density of solid phase . Then the volume fraction () of the solid phase in magnetic fluid is(3) hysteresis loops at room temperature of the -fe3n nanoparticles under the field up to 10 koe (s = 83.5 emu / g) if the interaction between magnetic particles in a certain magnetic fluid is ignored, the saturation magnetization of a magnetic fluid should be considered as the sum of the magnetization of all the dispersed magnetic particles . Taking the density () as a variable, the value of the saturation magnetization (emu / g) for magnetic fluids can be expressed as:(4) where sand are the magnetization of the dispersed particles and magnetic fluid, respectively . Figure 6 gives the relations between the calculated volume fraction () of particles and the density of the magnetic fluid samples using eq . It can be noticed that the density of the fluid increases linearly with increasing of iron - nitride magnetic particles in the fluid . The measured magnetization and the calculated values using eq . 4 for the different volume fractions of the -fe3n - based magnetic fluids are also shown in fig . It can be seen that both the calculated and measured magnetizations increase with increasing of the particles in the fluid . At the low concentrations, the measured values are close to the calculated one . With the increasing volume fraction of the -fe3n particles, the measured the phenomenon may be caused by two reasons: (1) particle volume fraction, (2) particle size . In magnetic fluid, in addition to the hydrodynamic interaction, there exists the dipolar magnetic dipolar interactions between magnetic particles are neglected and the magnetic particles in the fluid feel only the external magnetic field fitting the hypothesis of eq . 4 . For higher concentrations, dipolar dipolar interaction of particles cannot be ignored and this kind of interaction may affect particles relative motion when magnetized . The interaction between magnetic dipoles increases with the increase of particle concentration and the magnetic moments of part of the particles that are not fully aligned with the magnetic field . The magnetic fluid samples are not yet saturated during the measurement . Due to the wide particle size distribution, thus, the super paramagnetic fraction present in the samples might reduce the magnetization since the magnetic measurements were performed at room temperature . The effect of particle volume fraction () on density and magnetization of magnetic fluids in this work, the size of -fe3n nanoparticles was tested by tem and xrd . Compared with the results, both of the methods are suitable to particle size testing within the experimental error . Stable -fe3n - based magnetic fluid was prepared by controlling the ratio of carrier liquid and the optimized volume ratio for synthetic oil (pao4) and surfactant was 4:1 . Both the measured and calculated magnetizations increase with increasing particle fraction in the fluid . With the increasing concentration of the -fe3n particles, the measured values gradually depart from the calculated magnetization, which is caused by the volume fraction and size of the particle agglomeration.
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Short - lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (sunct) was first described in 1978 with diagnostic criteria established in 2004 [1, 2]. If associated with other cranial autonomic symptoms or without both conjunctival injection and tearing, the syndrome is classified as suna (short - lasting unilateral neuralgiform headache attacks with autonomic features). Sunct and suna are classified as trigeminal autonomic cephalalgias along with cluster headaches, and paroxysmal hemicrania with all of these headache syndromes sharing unilateral headache pain in a v1 trigeminal distribution and ipsilateral activation of cranial parasympathetics . Sunct has been reported in association with acquired and congenital abnormalities of the brainstem and pituitary region . We report a case of sunct with onset in adolescence in a patient with unilateral optic nerve hypoplasia (onh) and mild hypothalamic - pituitary dysfunction . A 27-year - old caucasian woman with a history of left onh diagnosed before 2 years of age was seen for evaluation of brief head pains . These head pains occurred over the right orbital region and lasted for around 30 s and rarely for more than a minute . The right eye became injected with tearing and right eyelid edema also occurred during the headaches . The patient would have this pattern of headaches 23 times per year with no apparent seasonal or diurnal pattern . The patient reported having hyperprolactinemia but prolactin levels from adolescence were not available for our review . In adolescence, she was tried on bromocriptine that did not adequately control her galactorrhea, but she had not had galactorrhea over the preceding 3 years . The patient had elevated prolactin levels of 31.0 and 50.3 ng per milliliter (normal range 3.3426.72 ng / ml) drawn at our facility and a low level of luteinizing hormone 0.76 milli - international units per milliliter (normal range 1.2103.0 miu / ml depending on stage of menstrual cycle) with normal levels of follicle stimulating hormone, renin, aldosterone, growth hormone, insulin - like growth factor-1, adrenocorticotrophic hormone, and thyroid stimulating hormone . Beginning in adolescence, the patient had spells of brief confusion and autonomic symptoms felt to be related to hypoglycemia with finger stick blood glucose levels of 4050 during or immediately after these spells . The patient had a personal history of simple febrile seizures occurring before age 2 with a family history of simple febrile seizures in her father, twin brother, and in two of her children . Neurologic examination was remarkable for strabismus and visual function limited to light perception in the left eye . Mri of the brain with and without contrast revealed a hypoplastic left optic nerve (fig . 1). An mri of the sellar region with contrast revealed an abnormality that followed the signal of cerebrospinal fluid on t1 and t2 sequences without complete attenuation on flair sequences consistent with a pituitary cyst (fig . 1). A seizure protocol mri brain did not reveal any abnormalities suggestive of a malformation of cortical development . No abnormalities of the meckel s cave region or posterior fossa were noted on any of these studies . . 1a coronal t2 sequence and b coronal flair sequence showing a small right pituitary cyst and normal septum pellucidum . D coronal orbital inversion recovery sequence showing left optic nerve hypoplasia a coronal t2 sequence and b coronal flair sequence showing a small right pituitary cyst and normal septum pellucidum . C coronal t1 sequence with gadolinium showing a normal pituitary gland and infundibulum . D coronal orbital inversion recovery sequence showing left optic nerve hypoplasia the patient has been followed for 8 months and continues to have a similar headache pattern . The patient s headaches are infrequent and unpredictable and determining an appropriate treatment strategy has been difficult . The headaches do not occur frequently enough to require preventive therapy . The patient is hesitant to start anti - epileptic medications such as topiramate or lamotrigine, so we prescribed indomethacin 25 mg to be used up to three times per day during symptomatic periods with the goal of reducing headache frequency and headache days . The patient s headache syndrome met ichd-2 criteria for short - lasting, unilateral neuralgiform headaches with conjunctival injection and tearing (sunct), and was consistent with more recent descriptions of this syndrome [13]. This patient did not report cutaneous or other triggering factors as seen in many sunct patients although spontaneous attacks may be present in 1428% of sunct patients . The clinical features were not suggestive of short - lasting paroxysmal hemicrania or trigeminal neuralgia . Sunct has been shown to have increased activation in the hypothalamus ipsilateral to the symptoms and in another case bilaterally on functional mri [4, 5]. There is also a case of sunct responding to hypothalamic stimulation ipsilateral to the symptoms and a case of a sunct patient responding to treatment with clomiphene citrate presumably due to its effects on the hypothalamus . There are numerous cases of sunct occurring in patients with prolactinomas sometimes during treatment with cabergoline or bromocriptine [813]. Sunct has also been in two patients with congenital bone abnormalities, basilar impression and craniosynostosis, with presumed involvement of the posterior fossa . Sunct typically has age of onset between 35 and 65 years with a mean around age 50 although ranges of age 1077 have been reported [2, 3]. Sunct associated with pituitary or brainstem abnormalities may have a slightly earlier age at onset with a mean around age 43 in one review . The case of craniosynostosis had onset of sunct at age 14 similar to the age of onset in our patient suggesting that congenital abnormalities of the posterior fossa or hypothalamic - pituitary axis may precipitate an earlier age of onset . Hypothalamic - pituitary dysfunction occurs in 75% of patients with onh and hyperprolactinemia occurs in 62% of these patients . The pituitary gland is abnormal in only 13% of patients with onh and the septum pellucidum is absent in 38% of patients . There may be a higher risk of endocrine dysfunction in onh patients with an absent septum pellucidum and abnormal pituitary gland on neuroimaging but this is not absolute as patients without these imaging features can still have hypothalamic - pituitary dysfunction . Patients with unilateral onh are at lower risk of hypothalamic - pituitary dysfunction than those with bilateral disease although dysfunction occurs in 69% of patient s with unilateral onh (compared to 81% with bilateral onh). Our patient s chronic hyperprolactinemia, abnormal menstrual cycles, and presumed hypoglycemic episodes were suggestive of mild hypothalamic - pituitary dysfunction . We were able to document hyperprolactinemia and low levels of luteinizing hormone, which were the likely associated with her history of abnormal menstrual cycles and galactorrhea . The strong personal and family history of simple febrile seizures may indicate a dysfunction of temperature regulation although another heritable cause cannot be excluded . Problems with body temperature regulation have been reported in onh, presumably due to hypothalamic dysfunction, with these patients sometimes requiring hospitalizations to rule out infections . Growth hormone deficiency and adrenocorticotrophic hormone deficiency are among the most common endocrinopathies in onh patients, both of which can result in hypoglycemia . We were not able to document a deficiency of growth hormone or adrenocorticotrophic hormone to explain the episodes of presumed hypoglycemia . The imaging characteristics were most consistent with a pituitary cyst and there was no other abnormality of her sellar region . We felt the pituitary cyst was most likely an incidental finding and not the likely cause of her mild hypothalamic dysfunction and sunct headaches . To our knowledge, this is the first reported case of sunct in a patient with onh . We hypothesize that there is an association between sunct headaches and the mild, hypothalamic - pituitary dysfunction in our patient . Congenital abnormality of the hypothalamic - pituitary axis may also explain the onset of this headache syndrome in adolescence.
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At the 2011 annual business meeting of the canadian geriatrics society (cgs), an ad hoc work group (wg) was struck and asked to submit a report at the time of the 2012 annual meeting of the cgs on physician resource needs in geriatrics . A voluntary lead for each province and territory was to be found who would be primarily responsible for their provincial / territorial data . As no funding or resources were allocated to this exercise, the wg was to use available data, wherever possible . The goal was not to create a definitive physician resource plan, but to provide an estimate of the number of physicians and full - time equivalents (ftes) currently working in the field of geriatrics, an estimate of the number required (if possible), and a clearer understanding of what has to be done to move physician resource planning in geriatrics forward . We do not address physician resource needs for consultants in geriatric psychiatry, medical directors in long - term care, physicians working in geriatric programs who do not provide consulting services (e.g., attending physician on in - patient geriatric unit), or primary medical care of older persons . Most older persons are (and will be) looked after by physicians without advanced clinical training in geriatrics . As such, all canadian physicians, except for those restricting their practice to fields where they will not encounter older individuals, must master the basic knowledge, skills, and attitudes required to provide effective care to older patients . Part of their responsibilities is to provide the instruction required to meet the educational needs of physicians without advanced clinical training in geriatrics . Relevant publications known to the authors were reviewed in the creation of this report (see reference list). Volunteer leads were identified for all provinces and territories (see list of authors; while in the main self - explanatory, dr . Teleconferences of the wg were held in the fall of 2011 to obtain input from members about the project and to seek agreement on core issues (e.g., scope of project, definition of specialist physicians in geriatrics), work plan, and timeline . It was agreed that we would focus on the need for specialist physicians in geriatrics . There was consensus that we would define specialist physicians in geriatrics as those who have completed advanced clinical training (or have equivalent work experience) in geriatrics and who limit a significant portion of their work - related activities to the duties of a consultant (defined as an individual to whom one refers for expert advice or services) in geriatrics . A preliminary list of specialist physicians in geriatrics (by name and location) michael borrie using data derived from the 2011 print edition of the canadian medical directory (scott s directories), the canadian medical directory online (scott s directories), the royal college of physicians and surgeons of canada (rcpsc) website, and the college of physicians and surgeons of ontario database . Provincial and territorial leads were asked to: verify that those listed for their province or territories (if any) were working as specialist physicians in geriatrics using the agreed upon definition;if known, indicate whether they were internists with rcpsc - accredited training in geriatric medicine, family physicians with college of family physicians of canada (cfpc)-accredited care of the elderly training, or had a different educational background (termed other);if known, note whether they were working part - time (which included those not working full - time and full - time workers who spent a substantial portion of their time in non - geriatric activities) or full - time in geriatrics (accepted work - related activities included administration and academic ones, as well as clinical work); for those working part - time, the leads were asked to provide a fte estimate of the time spent in geriatrics; and, identify physicians in their province or territory not listed who met the definition for specialist physicians in geriatrics (the leads were asked to provide the additional information noted above on them, if known). Verify that those listed for their province or territories (if any) were working as specialist physicians in geriatrics using the agreed upon definition; if known, indicate whether they were internists with rcpsc - accredited training in geriatric medicine, family physicians with college of family physicians of canada (cfpc)-accredited care of the elderly training, or had a different educational background (termed other); if known, note whether they were working part - time (which included those not working full - time and full - time workers who spent a substantial portion of their time in non - geriatric activities) or full - time in geriatrics (accepted work - related activities included administration and academic ones, as well as clinical work); for those working part - time, the leads were asked to provide a fte estimate of the time spent in geriatrics; and, identify physicians in their province or territory not listed who met the definition for specialist physicians in geriatrics (the leads were asked to provide the additional information noted above on them, if known). Based on the above, a final list of specialist physicians in geriatrics (by name and contact information with year of graduation [in order to estimate pending retirements], type of training, and fte, if known) was created . The leads were asked if there was a physician resource plan in their jurisdiction dealing with specialist physicians in geriatrics . A final teleconference was held in march of 2012 to discuss the initial results and their implications . A preliminary report was written by one of the co - chairs (dh) and distributed to wg members for review . Modifications to the document were made until agreement was obtained from wg members on the format and content of the report . Those identified in standard canadian references on the number of physicians in a specialty are nearly always certified specialists (i.e., physicians who have received certification from either the rcpsc or the collge des mdecins du qubec). We collated and summarized data on certified specialists in geriatric medicine from a number of standard sources (see table 1). There is a good deal of provincial and territorial variability . Excluding the three territories and using the maximum number of specialists reported, the ratio of certified specialists per 10,000 persons 65 + ranged more than ten - fold, from 0.07 (in saskatchewan) to 0.73 (nova scotia). The national ratio was 0.50 per 10,000 persons 65 + . While the number of certified specialists is useful information, there are limitations to this metric in assessing physician resources . A count of functional specialists is more appropriate, as the scope of physician practice is not strictly dictated by their certification . Not all certified specialists are active in their area of certification, while family practitioners and other physicians (often with additional training) may be providing care in fields such as geriatrics . The count of functional specialists can be either greater or less than the number of certified specialists, depending on the balance between non - certified physicians providing specialty care and certified specialists not providing care in their field of certification . Another issue is this is a head count and does not adjust for variations in the pattern or intensity of medical practice among individual physicians . Some of those counted may be semi - retired, working part - time, and/or providing a mix of medical services . Measuring physician resources by estimating ftes is an attempt to standardize physician activity to a common denominator based on output or productivity . The fte result for an individual physician quantifies their practice activity relative to what is considered a full load . In one, physician output / productivity is based on fee - for - service billings . The second uses multiple methodologies to measure the activities of physicians remunerated under alternative payment schemes . The last approach looks at hours of work per week . With the already significant and growing number of physicians in geriatrics being paid at least partially through alternative payment plans, methodologies for determining ftes in this field will have to be flexible enough to collect data on all relevant activities being provided regardless of the mode of payment . Ideally, measures of productivity should include an assessment of the quality of service provided and patient outcome data . With the information provided by wg members, estimates for the number and ftes of functional specialists in geriatrics were determined (see table 2). As noted previously, in calculating ftes administrative, academic (teaching and research), and clinical activities were included . The reasons for this include the general definition used for specialist physicians in geriatrics (meaning judgment had to be exercised in deciding who to include), the lack of a clear description of a fte in geriatrics, the need to rely on the personal knowledge of provincial / territorial leads about local physician resources and/or their collection of primary data, difficulties obtaining confirmatory information, and limited resources and time . More precise data would require clearer definitions for both a specialist and an fte, consistently adhering to these definitions in determining ftes, and systematically obtaining primary information . The latter would require a substantial investment on the part of the cgs and/or others . Notwithstanding this, we feel the data presented represents an important advance in understanding current physician resources . While the total number of physicians providing consultative services in geriatrics (n = 404) is substantially higher than the number of certified specialists (n = 230242), it is reassuring that the number of internists with rcpsc - accredited training in geriatric medicine in table 2 is congruent with the totals the workforce consists of a mix of certified / non - certified and part - time / full - time specialists . Even if desired, it would not be possible within the foreseeable future to produce a sufficient number of certified specialists to deal with current and anticipated demand . Other than inadequate supply and personal choice about where to work and live, factors that limit a wider and more even distribution of certified specialists include insufficient population of older persons and/or inadequate resources for a specialty practice in geriatrics within given areas, the health needs of the local population (which will influence the priority given to geriatrics in comparison to other medical fields when recruiting physicians), and financial constraints (e.g., funding for required infrastructure, availability of billing numbers and/or salary support for physicians). Data on the reasons why physicians with accredited training chose to work part - time in geriatrics were not collected but could include income (i.e., spending time in a more remunerative field to bolster income), opportunities for more contact with medical students and postgraduate trainees (e.g., doing rotations as staff physician on medical teaching units), employment requirements (e.g., contractual obligation to provide general medical services), and/or personal interest . We examined emergency and palliative medicine, which are areas of specialty practice in canada facing similar challenges in merging the activities of physicians with different educational backgrounds within the workplace, to learn from their approaches . There are two routes for training in emergency medicine in canada a five - year residency program accredited by the rcpsc, and a one - year training program after a two - year family medicine residency through the cfpc . Most full - time emergency department (ed) physicians in our country are now certified through one of these two routes . Cfpc - certified ed physicians outnumber rcpsc ones by a ratio of about three to one and work primarily as clinicians . In the 2012 family medicine emergency medicine match, a total of 128 training positions were filled . The majority of family physicians with an emergency medicine certification work primarily in eds . Rcpsc - certified physicians tend to congregate in academic centres and are more likely to do non - clinical work (e.g., administration, teaching, research, disaster planning, advising on pre - hospital care, and toxicology). Based on the canadian medical association (cma) masterfile on physicians, as of 2012 there were 667 certified specialists in emergency medicine . In 201011, there were a total of 294 residents enrolled in rcpsc - accredited emergency medicine training programs (approximately 59 per year). The two training options are felt to complement each other . At a time when there are shortages in physicians with training in emergency medicine, a leader of the field wrote that it does not make sense to restrict clinical positions to rcpsc - accredited graduates . The rcpsc accredits palliative medicine training programs, but also does not offer a certifying examination (the rcpsc is phasing out this type of training program). Graduates receive an attestation of completion of training from their respective colleges . Between 1999 and 2010, 75 trainees were attested by the cfpc and 21 by the rcpsc . In 201011, there were 34 residents enrolled in a palliative medicine training program . In the future, it is proposed that a two - year subspecialty training program will be offered through the rcpsc, while a one - year program leading to a special designation in palliative medicine will be available under the aegis of the cfpc . It appears that the field is moving towards a model similar to emergency medicine where cfpc - accredited training will prepare physicians for clinical work, while those trained through a rcpsc program will presumably focus more on administration and academic pursuits . There were 311 members of the canadian society of palliative care physicians . Within the cfpc there continues to be an energetic debate about whether palliative care should be a specialty or considered a core component of family medicine . In order to address the number of specialist physicians in geriatrics needed now and in the near future, we collected information on physician resource plans for specialist physicians in geriatrics created by provinces, territories, and/or health regions . As will be seen, most jurisdictions have not developed comprehensive physician resource plans for geriatrics . There are no physician resource plans for geriatrics in any of the three territories or the provinces of british columbia (though most, but not all, health authorities have physician resource plans, with some actively recruiting), alberta (local physician resource plans have been created for the alternate relationship plans of the departments of medicine in calgary and edmonton), saskatchewan, manitoba (the section of geriatric medicine at the university of manitoba believes that 10 ftes in geriatrics are required), or qubec . In ontario the five regional geriatric programs and affiliated specialized geriatric services (sgs) have initiated a mapping process for each of the local integrated health networks (lhins) that compares the number of known practicing internist geriatricians, cfpc care of the elderly trained physicians, and other family physicians working in sgs by lhin with the estimated required number for the lhin calculated using a ratio of 1.25 geriatricians per 10,000 people over the age of 65 . The surplus or deficit for the lhin is then determined . The ministry of health and long - term care and ontario medical association s 20082012 agreement endorsed up to 10 new geriatrician positions . A larger number of new positions are being requested in current negotiations . There is a provincial resource plan in new brunswick for the total number of physicians, but no target exists for geriatricians . Vacant and new positions for physicians are assigned to the eight health zones of that province . Within zones there are discussions about which specialties should be recruited to fill vacancies . Until now, it was been possible to successfully argue for one of these vacancies when there was a firm (person on the ground) the environment is changing, and it is getting more difficult to recruit . In p.e.i ., the provincial ministry of health has identified a need for three specialists in geriatrics . The dalhousie division of geriatric medicine, with input from the health districts in nova scotia, has developed a provincial workforce plan for geriatrics . In newfoundland and labrador, physician resources are planned using a physician / population ratio model (e.g., so many per current population). There is a recognized need for three rcpsc certified specialists in geriatric medicine, but currently there is no target for the number of care of the elderly physicians required . A commonly used approach in assessing current and future physician resource need are physician / population ratios . In 198889, it was estimated that 131 specialists in geriatrics were needed for the province of ontario . Though not stated, it was implied that all these physicians would be working full - time in geriatrics (i.e., would be 1.0 fte). Ninety - five were for clinical service, 29 to meet academic need, and seven for administration . A questionnaire was sent to 19 persons considered experts in health human resource planning, requesting their views on the roles of specialist physicians in geriatric medicine . Attached to the questionnaire participants were asked to provide estimates for the number of specialists in geriatric medicine required for clinical service, academic roles, and administration in these regions . Their estimates were tabulated and returned to responders asking them to either accept or revise the estimates . Eleven provided further input (recipients were told that non - return would indicate agreement). After incorporating this feedback, one of the 14 declined to approve the final estimates, feeling they were too high . This study is the source for the oft - quoted ratio of 1.25 specialists in geriatrics per 10,000 individuals 65 + (or 3.3 per 10,000 individuals 75 +). The current ratio in canada is between 0.48 (based on number of certified specialists) and 0.84 (based on number of functional specialists) per 10,000 persons 65 + . Using this approach, it was estimated that there was a need for 538 specialists in geriatric medicine in 2006 . This number was referred to by the national advisory council of aging and the special senate committee on aging . Relying on a ratio for work force planning is imprecise as it does nt accurately reflect actual need, adjust for variations in the intensity of medical practice, or acknowledge the role of other providers . This particular ratio is based on the opinion of a small group from one province obtained nearly a quarter of a century ago . As a minimum, imported ratios from other countries are suspect as they do nt account for international differences in factors such as population structure and expectations, practitioner occupational niche(s), structure and function of the health - care system, and/or availability of health - care resources . In the united kingdom (uk), the royal college of physicians (rcp) recommends six fte trained specialists in geriatric medicine per 250,000 (total population) or approximately one per 40,000 persons . The british geriatrics society accepts this figure and also suggests there be one fte per 4,000 persons over 75 . Aside from other differences between canada and the uk, the roles of geriatric specialists in the two countries are not identical . Compared to canadian practitioners, uk geriatricians are more heavily committed to acute, general, and stroke medicine but less to dementia care . In 2010 there were 980 consultants in geriatric medicine (941 ftes) employed by the national health service in england . There are now four general approaches used to model and forecast physician supply and demand: supply forecasting, utilization or demand forecasting, needs - based planning, and benchmarking . Supply forecasting looks at the number of physicians (of a particular type) anticipated to enter the work force (see future influences on physician supply as models become more complex, they are increasingly affected by data limitations, and become more dependent on judgment and assumptions of stability . Benchmarking avoids assumptions of stability in the health - care system . It identifies areas where delivery is perceived to be optimal and uses them as the standard . Rigorous utilization and demand forecasting, needs - based planning, and/or benchmarking have not been used to develop a national physician resource plan in geriatrics in our country . For one thing, there are no conditions, procedures or age ranges that fall solely within the domain of specialists in geriatrics . As noted, the majority of older persons are (and will be) looked after by physicians without advanced clinical training in geriatrics . Older patients who are felt to require the input of geriatric specialists (or specialized geriatric services) are those with complex issues, cognitive impairment, disability, and/or frailty . More precisely defining who they are, when they should be referred, where they should be seen, and for what type of service (and for how long) has not, by and large, taken place . Requests for assistance in the assessment and management of dementia, the most common condition treated by canadian geriatricians, could be directed to neurologists and geriatric psychiatrists / psychiatrists, as well as geriatricians, while improving the abilities of primary care physicians to deal with this clinical condition would partially obviate the need for referrals in the first place . Compared to many other fields, though, there is an extensive body of research showing the benefits of care provided by geriatric specialists and the programs they work in . This can be used to provide a clearer picture of the desired role (and size) for the specialty . They are more likely to be part of an interdisciplinary team and have lower patient volumes (the reported mean number of estimated patient visits during a typical week for a specialist in geriatric medicine was 38.81 in the 2004 national physician survey compared to 67.29 for general internists), presumably because of the nature of patients seen and the type of care provided . The closest we ever came to a national physician resource plan in geriatrics was a 1988 estimate based on a survey of national leaders that we would require 550700 specialists in geriatric medicine by 2000 . An interesting but now older study estimated american physician resource needs in geriatrics . Based on medical service utilization, physician clinical workloads in the mid-1980s, and perceived need for geriatricians, it looked at requirements for both primary and consultant care within three different scenarios reflecting different economic assumptions . The projected required fte number for primary and consultant care combined ranged from 3,153 to 12,057 . In the united states, geriatrics recent estimates for the number of ftes in geriatrics required by 2030 have risen substantially, to between 26,000 and 36,000 . These are based on assumptions about the proportion of older persons who should be cared for by geriatricians (i.e., 2530%) and the number of older patients a geriatrician can effectively deal with . The wide range gives a sense of the difficulty in coming up with accurate figures and how sensitive estimates are to underlying assumptions . In 2010 the number of active physicians in canada is increasing at a faster rate than population growth . Over time, the average age of the physician work force has risen (it is now 50), as has the proportion of females (from 12.4% in 1980 to 36.1% in 2010). One hundred and twenty - nine of the 237 (54.4%) certified canadian specialists in geriatrics where gender was known and 18 of the 30 (60%) current postgraduate trainees in geriatrics are female . Rural and remote areas have particular challenges in accessing specialty services . Compared to older practitioners, younger physicians want to work fewer (and more predictable) hours within group rather than solo practices . Family medicine residents and cfpc program directors feel that approximately one - third of core family medicine program graduates should have the opportunity of a third year of training . Changes in educational policy (e.g., eligibility requirements, standards of accreditation, certifying examination) by the cfpc and/or rcpsc could have significant impacts on future supply . It is assumed that those reading this report are knowledgeable about rcpsc - accredited training in geriatric medicine and cfpc - accredited training in care of the elderly . We are unaware of any current proposal for substantive change to either training program . In the past, alternate approaches to postgraduate training were considered, though nothing came of them . In 1976, the rcpsc committee on specialty development and manpower suggested that entry for training in geriatric medicine be open to those with prior certification in specialties other than internal medicine . Ten years later, the cma committee on the health care of the elderly recommended that geriatric medicine become a primary specialty and that a mechanism for recognizing prior cfpc training as partial requirement for certification be worked out . Areas of focused competence (or diploma) category will allow the rcpsc to recognize highly focused areas of specialty medicine, while special interest groups of medical activity will serve as forums for individuals with common areas of interest . Neither designation is to adversely impact existing disciplines, but it is unclear at this time what, if any, effect this will have on rcpsc - accredited training in geriatric medicine . The potential impact of sub - specialization within geriatrics (e.g., dementia care) on physician resources was not considered by the wg . Supply - side approaches to deal with the perceived inadequate number of specialist physicians in geriatrics are described in the following sections . The total number of residents enrolled in a program of advanced clinical training in geriatrics over the last ten years has been limited . While the numbers vary from year - to - year, there is no clear evidence of growth (see table 3). In 201011 there were 30 (11 care of the elderly trainees and 19 enrolled in geriatric medicine). Training locations (which indicate where they are more likely to practice upon completion of their training) for the 201011 cohort of residents are: 16 in ontario (12 toronto, 1 mcmaster, 3 uwo); 5 british columbia; 4 qubec (2 laval, 2 mcgill); 3 prairies (1 manitoba, 2 university of alberta); and 2 in atlantic canada . The estimated entering practice cohort of physicians with additional training in geriatrics in 2011 was 17 (total of nine r3 care of the elderly and eight r5 geriatric medicine trainees). Over the last five years, the entering practice cohort has ranged from 17 to 26, which is too small to substantially change access to consultants in geriatrics, especially when one considers pending retirements, challenges in retention, and the rapidly increasing number of older canadians that will be seen over the next 30 years . The primary problem is not having a sufficient total number of positions for qualified residents applying for training in geriatric medicine, as more than half of all available training positions have been unfilled recently . In the 2012 carms r4 medicine subspecialty match, less than half (42.3%, 11/26) of residency positions offered were filled . These rates are lower than those seen for the larger sub - specialties of internal medicine . This is not to say that there are no concerns about the distribution of available positions, but the total number of training positions is more than adequate to meet current demand . Postgraduate training in geriatric medicine has become concentrated in two faculties the university of toronto and ubc . Over the last three years, 19 out of the 32 (59.4%) residents who entered a training program in geriatric medicine one would expect this concentration to continue . As residents entering practice tend to stay close to where they received their training, this could present recruitment challenges to other regions of the country . The situation might well change in the future . For one thing, there is reawakening interest in developing a national strategy to better align physician training to population needs . Employment difficulties for new graduates have been reported in seven specialties (cardiac surgery, nephrology, neurosurgery, plastic surgery, public health and preventive medicine [community medicine], otolaryngology, radiation oncology) with other fields (e.g., orthopedic surgery) expected to soon have similar challenges . Students may vote with their feet, with fewer applying for training in these fields . Students are asking for better career planning tools . A motion passed at the 2011 cma annual meeting called upon the organization to provide job - trend data and other career planning resources . Another resolution will compel the association to promote a national alignment of training positions with current and future societal needs, evolving models of care delivery and available health system resources . More effective marketing of geriatrics to both medical students and postgraduate trainees might bear fruit . More re - entry training positions for cfpc and rcpsc - accredited training would allow greater opportunities for practicing physicians to re - tool as specialists in geriatrics . This would require funding and the allocation of residency positions . A number of the functional specialists listed in table 2 were trained on the job . Standards and educational resources need to be developed for them . Allowing more international medical graduates with geriatric training to enter we would need to ensure that the training and skills they bring are congruent with our national needs, they remain in geriatrics after moving to canada, and their recruitment does nt disadvantage canadian trainees . There are ethical concerns about recruiting internationally - trained health professionals from developing countries that also require consideration . Having trained individuals leave geriatrics is a regrettable and potentially preventable loss of physician resources . Retention appears to be a particular issue for those with care of the elderly training . A survey of care of the elderly graduates indicated that over half (55.8%) end up in a general family medicine practice . Since the development of this postgraduate training option in 1989, an estimated 160170 physicians (the cfpc does nt have a precise cumulative count) have completed training . Based on our determination of functional specialists, an estimated 40 of these physicians is examining the time spent in other clinical areas by practicing geriatricians . Among rcpsc - accredited specialists we need to assess the relative benefits and negative consequences of this on specialized geriatric services in our country . For example, specialists in geriatric medicine often attend on medical teaching units and/or work as internists in emergency rooms . In addition to providing a greater opportunity for them to instruct medical students and postgraduate trainees on the principles of geriatric care, there could be direct benefits for the many older patients they would see . Because of the unique knowledge, skill set, and approach they bring to the care of these patients, their involvement might lead to better outcomes such as lower mortality rates, better functional outcomes, less iatrogenesis, a greater proportion discharged back home, and/or lower readmission rates (these assertions could be evaluated in a pragmatic clinical trial). We have to weigh, though, the opportunity cost (i.e., the benefit that could have arisen by taking alternative action) of the time spent in non - geriatric activities and the risk of spreading ourselves too thin, which could have negative impacts on both the quality of work provided and its long - term sustainability . With our limited physician resources, we have to carefully prioritize the various ways we allocate our time . Concurrently we should point out to decision - makers the additional meritorious activities we could perform if we had sufficient numbers . According to the cma masterfile, 82 of the 233 (35.2%) certified specialists in geriatrics where age is known are 55 years of age and older . While hard to precisely predict when a given physician will retire, many practicing specialist physicians are closer to the end of their careers than to the beginning . If one assumes that physicians retire 40 years after receiving their md degree, we can estimate the expected number of retirements over the coming years . We should not focus solely on the complete cessation of work as prior to this, many physicians will reduce their workload and/or narrow their scope of practice, which has implications for physician work force planning . Strategies to keep older physicians engaged would include creating more job opportunities in non - clinical areas and flexibility in structuring part - time work . This is critical in all supply - side attempts to deal with a deficient number of practitioners . While efforts to improve physician recruitment and retention have historically focused on financial incentives, other ones should be considered . The overlapping factors that influence career decisions can be categorized as educational (e.g., early and on - going exposure to the field, length and availability of training opportunities, exposure to role models / mentors), professional (e.g., availability of positions, scope and type of practice, workload, frequency and intensity of on call responsibilities, professional support and collegiality, administrative support and availability of infrastructure, opportunities for professional development), personal or lifestyle (e.g., pre - existing interests and life experiences, desire for work - life balance, opportunities for part - time work / job sharing, family - friendly work environment, quality of life), and financial (e.g., level and stability of income, reimbursement for overhead expenses, availability of bursaries, debt relief / assistance, student loan forgiveness). A recent report by the institute for clinical evaluative sciences showed that mean and median payments to geriatricians remained significantly below the average for all physicians in ontario . (note: at the time this report was written, similar data were not available for other provinces .) Poor remuneration has long been perceived by many geriatricians as a major barrier to recruitment . While physicians should not be bribed to work in geriatrics, remuneration should not be a disincentive to considering the field . To make serious inroads on these issues will require a long - term commitment on the part of practitioners, medical and health leaders, and the cgs . Our ultimate goal is to improve the accessibility, effectiveness, and efficiency of specialized geriatric services . The first goal in particular might benefit from the innovative use of technology (e.g., telehealth, decision - support information technology) and labour substitution (e.g., expanding scopes of practice for other health professionals). Specialist physicians in geriatrics require infrastructure (e.g., interdisciplinary teams) to be fully effective . It is important to ensure that these teams are funded and available to them . Over time, new approaches and alternative models of care for older patients will be developed . As a specialty we should support their creation, critical evaluation, and dissemination, if found to be worthwhile . Those identified in standard canadian references on the number of physicians in a specialty are nearly always certified specialists (i.e., physicians who have received certification from either the rcpsc or the collge des mdecins du qubec). We collated and summarized data on certified specialists in geriatric medicine from a number of standard sources (see table 1). There is a good deal of provincial and territorial variability . Excluding the three territories and using the maximum number of specialists reported, the ratio of certified specialists per 10,000 persons 65 + ranged more than ten - fold, from 0.07 (in saskatchewan) to 0.73 (nova scotia). The national ratio was 0.50 per 10,000 persons 65 + . While the number of certified specialists is useful information, there are limitations to this metric in assessing physician resources . A count of functional specialists is more appropriate, as the scope of physician practice is not strictly dictated by their certification . Not all certified specialists are active in their area of certification, while family practitioners and other physicians (often with additional training) may be providing care in fields such as geriatrics . The count of functional specialists can be either greater or less than the number of certified specialists, depending on the balance between non - certified physicians providing specialty care and certified specialists not providing care in their field of certification . Another issue is this is a head count and does not adjust for variations in the pattern or intensity of medical practice among individual physicians . Some of those counted may be semi - retired, working part - time, and/or providing a mix of medical services . Measuring physician resources by estimating ftes is an attempt to standardize physician activity to a common denominator based on output or productivity . The fte result for an individual physician quantifies their practice activity relative to what is considered a full load . In one, physician output / productivity is based on fee - for - service billings . The second uses multiple methodologies to measure the activities of physicians remunerated under alternative payment schemes . The last approach looks at hours of work per week . With the already significant and growing number of physicians in geriatrics being paid at least partially through alternative payment plans, methodologies for determining ftes in this field will have to be flexible enough to collect data on all relevant activities being provided regardless of the mode of payment . Ideally, measures of productivity should include an assessment of the quality of service provided and patient outcome data . With the information provided by wg members, estimates for the number and ftes of functional specialists in geriatrics were determined (see table 2). As noted previously, in calculating ftes administrative, academic (teaching and research), and clinical activities were included . The reasons for this include the general definition used for specialist physicians in geriatrics (meaning judgment had to be exercised in deciding who to include), the lack of a clear description of a fte in geriatrics, the need to rely on the personal knowledge of provincial / territorial leads about local physician resources and/or their collection of primary data, difficulties obtaining confirmatory information, and limited resources and time . More precise data would require clearer definitions for both a specialist and an fte, consistently adhering to these definitions in determining ftes, and systematically obtaining primary information . The latter would require a substantial investment on the part of the cgs and/or others . Notwithstanding this, we feel the data presented represents an important advance in understanding current physician resources . While the total number of physicians providing consultative services in geriatrics (n = 404) is substantially higher than the number of certified specialists (n = 230242), it is reassuring that the number of internists with rcpsc - accredited training in geriatric medicine in table 2 is congruent with the totals noted in table 1 . The workforce consists of a mix of certified / non - certified and part - time / full - time specialists . Even if desired, it would not be possible within the foreseeable future to produce a sufficient number of certified specialists to deal with current and anticipated demand . Other than inadequate supply and personal choice about where to work and live, factors that limit a wider and more even distribution of certified specialists include insufficient population of older persons and/or inadequate resources for a specialty practice in geriatrics within given areas, the health needs of the local population (which will influence the priority given to geriatrics in comparison to other medical fields when recruiting physicians), and financial constraints (e.g., funding for required infrastructure, availability of billing numbers and/or salary support for physicians). Data on the reasons why physicians with accredited training chose to work part - time in geriatrics were not collected but could include income (i.e., spending time in a more remunerative field to bolster income), opportunities for more contact with medical students and postgraduate trainees (e.g., doing rotations as staff physician on medical teaching units), employment requirements (e.g., contractual obligation to provide general medical services), and/or personal interest . We examined emergency and palliative medicine, which are areas of specialty practice in canada facing similar challenges in merging the activities of physicians with different educational backgrounds within the workplace, to learn from their approaches . There are two routes for training in emergency medicine in canada a five - year residency program accredited by the rcpsc, and a one - year training program after a two - year family medicine residency through the cfpc . Most full - time emergency department (ed) physicians in our country are now certified through one of these two routes . Cfpc - certified ed physicians outnumber rcpsc ones by a ratio of about three to one and work primarily as clinicians . In the 2012 family medicine emergency medicine match, a total of 128 training positions were filled . The majority of family physicians with an emergency medicine certification work primarily in eds . Rcpsc - certified physicians tend to congregate in academic centres and are more likely to do non - clinical work (e.g., administration, teaching, research, disaster planning, advising on pre - hospital care, and toxicology). Based on the canadian medical association (cma) masterfile on physicians, as of 2012 there were 667 certified specialists in emergency medicine . In 201011, there were a total of 294 residents enrolled in rcpsc - accredited emergency medicine training programs (approximately 59 per year). The two training options are felt to complement each other . At a time when there are shortages in physicians with training in emergency medicine, the rcpsc accredits palliative medicine training programs, but also does not offer a certifying examination (the rcpsc is phasing out this type of training program). Graduates receive an attestation of completion of training from their respective colleges . Between 1999 and 2010, 75 trainees were attested by the cfpc and 21 by the rcpsc . In 201011, there were 34 residents enrolled in a palliative medicine training program . In the future, it is proposed that a two - year subspecialty training program will be offered through the rcpsc, while a one - year program leading to a special designation in palliative medicine will be available under the aegis of the cfpc . It appears that the field is moving towards a model similar to emergency medicine where cfpc - accredited training will prepare physicians for clinical work, while those trained through a rcpsc program will presumably focus more on administration and academic pursuits . There were 311 members of the canadian society of palliative care physicians . Within the cfpc there continues to be an energetic debate about whether palliative care should be a specialty or considered a core component of family medicine . In order to address the number of specialist physicians in geriatrics needed now and in the near future, we collected information on physician resource plans for specialist physicians in geriatrics created by provinces, territories, and/or health regions . As will be seen, most jurisdictions have not developed comprehensive physician resource plans for geriatrics . There are no physician resource plans for geriatrics in any of the three territories or the provinces of british columbia (though most, but not all, health authorities have physician resource plans, with some actively recruiting), alberta (local physician resource plans have been created for the alternate relationship plans of the departments of medicine in calgary and edmonton), saskatchewan, manitoba (the section of geriatric medicine at the university of manitoba believes that 10 ftes in geriatrics are required), or qubec . In ontario the five regional geriatric programs and affiliated specialized geriatric services (sgs) have initiated a mapping process for each of the local integrated health networks (lhins) that compares the number of known practicing internist geriatricians, cfpc care of the elderly trained physicians, and other family physicians working in sgs by lhin with the estimated required number for the lhin calculated using a ratio of 1.25 geriatricians per 10,000 people over the age of 65 . The surplus or deficit for the lhin is then determined . The ministry of health and long - term care and ontario medical association s 20082012 agreement endorsed up to 10 new geriatrician positions . A larger number of new positions are being requested in current negotiations . There is a provincial resource plan in new brunswick for the total number of physicians, but no target exists for geriatricians . Vacant and new positions for physicians are assigned to the eight health zones of that province . Within zones, it was been possible to successfully argue for one of these vacancies when there was a firm (person on the ground) the environment is changing, and it is getting more difficult to recruit . In p.e.i ., the provincial ministry of health has identified a need for three specialists in geriatrics . The dalhousie division of geriatric medicine, with input from the health districts in nova scotia, has developed a provincial workforce plan for geriatrics . In newfoundland and labrador, physician resources are planned using a physician / population ratio model (e.g., so many per current population). There is a recognized need for three rcpsc certified specialists in geriatric medicine, but currently there is no target for the number of care of the elderly physicians required . A commonly used approach in assessing current and future physician resource need are physician / population ratios . In 198889, it was estimated that 131 specialists in geriatrics were needed for the province of ontario . Though not stated, it was implied that all these physicians would be working full - time in geriatrics (i.e., would be 1.0 fte). Ninety - five were for clinical service, 29 to meet academic need, and seven for administration . A questionnaire was sent to 19 persons considered experts in health human resource planning, requesting their views on the roles of specialist physicians in geriatric medicine . Attached to the questionnaire was a map of the then five provincial health regions (with populations). Participants were asked to provide estimates for the number of specialists in geriatric medicine required for clinical service, academic roles, and administration in these regions . Their estimates were tabulated and returned to responders asking them to either accept or revise the estimates . Eleven provided further input (recipients were told that non - return would indicate agreement). After incorporating this feedback, one of the 14 declined to approve the final estimates, feeling they were too high . This study is the source for the oft - quoted ratio of 1.25 specialists in geriatrics per 10,000 individuals 65 + (or 3.3 per 10,000 individuals 75 +). The current ratio in canada is between 0.48 (based on number of certified specialists) and 0.84 (based on number of functional specialists) per 10,000 persons 65 + . Using this approach, it was estimated that there was a need for 538 specialists in geriatric medicine in 2006 . This number was referred to by the national advisory council of aging and the special senate committee on aging . Relying on a ratio for work force planning is imprecise as it does nt accurately reflect actual need, adjust for variations in the intensity of medical practice, or acknowledge the role of other providers . This particular ratio is based on the opinion of a small group from one province obtained nearly a quarter of a century ago . As a minimum, imported ratios from other countries are suspect as they do nt account for international differences in factors such as population structure and expectations, practitioner occupational niche(s), structure and function of the health - care system, and/or availability of health - care resources . In the united kingdom (uk), the royal college of physicians (rcp) recommends six fte trained specialists in geriatric medicine per 250,000 (total population) or approximately one per 40,000 persons . The british geriatrics society accepts this figure and also suggests there be one fte per 4,000 persons over 75 . Aside from other differences between canada and the uk, the roles of geriatric specialists in the two countries are not identical . Compared to canadian practitioners, uk geriatricians are more heavily committed to acute, general, and stroke medicine but less to dementia care . In 2010 there were 980 consultants in geriatric medicine (941 ftes) employed by the national health service in england . There are now four general approaches used to model and forecast physician supply and demand: supply forecasting, utilization or demand forecasting, needs - based planning, and benchmarking . Supply forecasting looks at the number of physicians (of a particular type) anticipated to enter the work force (see future influences on physician supply as models become more complex, they are increasingly affected by data limitations, and become more dependent on judgment and assumptions of stability . Benchmarking avoids assumptions of stability in the health - care system . It identifies areas where delivery is perceived to be optimal and uses them as the standard . Rigorous utilization and demand forecasting, needs - based planning, and/or benchmarking have not been used to develop a national physician resource plan in geriatrics in our country . For one thing, there are no conditions, procedures or age ranges that fall solely within the domain of specialists in geriatrics . As noted, the majority of older persons are (and will be) looked after by physicians without advanced clinical training in geriatrics . Older patients who are felt to require the input of geriatric specialists (or specialized geriatric services) are those with complex issues, cognitive impairment, disability, and/or frailty . More precisely defining who they are, when they should be referred, where they should be seen, and for what type of service (and for how long) has not, by and large, taken place . Requests for assistance in the assessment and management of dementia, the most common condition treated by canadian geriatricians, could be directed to neurologists and geriatric psychiatrists / psychiatrists, as well as geriatricians, while improving the abilities of primary care physicians to deal with this clinical condition would partially obviate the need for referrals in the first place . Compared to many other fields, though, there is an extensive body of research showing the benefits of care provided by geriatric specialists and the programs they work in . This can be used to provide a clearer picture of the desired role (and size) for the specialty . They are more likely to be part of an interdisciplinary team and have lower patient volumes (the reported mean number of estimated patient visits during a typical week for a specialist in geriatric medicine was 38.81 in the 2004 national physician survey compared to 67.29 for general internists), presumably because of the nature of patients seen and the type of care provided . The closest we ever came to a national physician resource plan in geriatrics was a 1988 estimate based on a survey of national leaders that we would require 550700 specialists in geriatric medicine by 2000 . An interesting but now older study estimated american physician resource needs in geriatrics . Based on medical service utilization, physician clinical workloads in the mid-1980s, and perceived need for geriatricians, it looked at requirements for both primary and consultant care within three different scenarios reflecting different economic assumptions . The projected required fte number for primary and consultant care combined ranged from 3,153 to 12,057 . In the united states, geriatrics recent estimates for the number of ftes in geriatrics required by 2030 have risen substantially, to between 26,000 and 36,000 . These are based on assumptions about the proportion of older persons who should be cared for by geriatricians (i.e., 2530%) and the number of older patients a geriatrician can effectively deal with . The wide range gives a sense of the difficulty in coming up with accurate figures and how sensitive estimates are to underlying assumptions . In 2010 the number of active physicians in canada is increasing at a faster rate than population growth . Over time, the average age of the physician work force has risen (it is now 50), as has the proportion of females (from 12.4% in 1980 to 36.1% in 2010). One hundred and twenty - nine of the 237 (54.4%) certified canadian specialists in geriatrics where gender was known and 18 of the 30 (60%) current postgraduate trainees in geriatrics are female . Compared to older practitioners, younger physicians want to work fewer (and more predictable) hours within group rather than solo practices . Family medicine residents and cfpc program directors feel that approximately one - third of core family medicine program graduates should have the opportunity of a third year of training . Changes in educational policy (e.g., eligibility requirements, standards of accreditation, certifying examination) by the cfpc and/or rcpsc could have significant impacts on future supply . It is assumed that those reading this report are knowledgeable about rcpsc - accredited training in geriatric medicine and cfpc - accredited training in care of the elderly . We are unaware of any current proposal for substantive change to either training program . In the past, alternate approaches to postgraduate training were considered, though nothing came of them . In 1976, the rcpsc committee on specialty development and manpower suggested that entry for training in geriatric medicine be open to those with prior certification in specialties other than internal medicine . Ten years later, the cma committee on the health care of the elderly recommended that geriatric medicine become a primary specialty and that a mechanism for recognizing prior cfpc training as partial requirement for certification be worked out . Areas of focused competence (or diploma) category will allow the rcpsc to recognize highly focused areas of specialty medicine, while special interest groups of medical activity will serve as forums for individuals with common areas of interest . Neither designation is to adversely impact existing disciplines, but it is unclear at this time what, if any, effect this will have on rcpsc - accredited training in geriatric medicine . The potential impact of sub - specialization within geriatrics (e.g., dementia care) on physician resources was not considered by the wg . Supply - side approaches to deal with the perceived inadequate number of specialist physicians in geriatrics are described in the following sections . The total number of residents enrolled in a program of advanced clinical training in geriatrics over the last ten years has been limited . While the numbers vary from year - to - year, there is no clear evidence of growth (see table 3). In 201011 there were 30 (11 care of the elderly trainees and 19 enrolled in geriatric medicine). Training locations (which indicate where they are more likely to practice upon completion of their training) for the 201011 cohort of residents are: 16 in ontario (12 toronto, 1 mcmaster, 3 uwo); 5 british columbia; 4 qubec (2 laval, 2 mcgill); 3 prairies (1 manitoba, 2 university of alberta); and 2 in atlantic canada . The estimated entering practice cohort of physicians with additional training in geriatrics in 2011 was 17 (total of nine r3 care of the elderly and eight r5 geriatric medicine trainees). Over the last five years, the entering practice cohort has ranged from 17 to 26, which is too small to substantially change access to consultants in geriatrics, especially when one considers pending retirements, challenges in retention, and the rapidly increasing number of older canadians that will be seen over the next 30 years . The primary problem is not having a sufficient total number of positions for qualified residents applying for training in geriatric medicine, as more than half of all available training positions have been unfilled recently . In the 2012 carms r4 medicine subspecialty match, less than half (42.3%, 11/26) of residency positions offered were filled . These rates are lower than those seen for the larger sub - specialties of internal medicine . This is not to say that there are no concerns about the distribution of available positions, but the total number of training positions is more than adequate to meet current demand . Postgraduate training in geriatric medicine has become concentrated in two faculties the university of toronto and ubc . Over the last three years, 19 out of the 32 (59.4%) residents who entered a training program in geriatric medicine were matched to these schools . In sub - specialty training, one would expect this concentration to continue . As residents entering practice tend to stay close to where they received their training, this could present recruitment challenges to other regions of the country . For one thing, there is reawakening interest in developing a national strategy to better align physician training to population needs . Employment difficulties for new graduates have been reported in seven specialties (cardiac surgery, nephrology, neurosurgery, plastic surgery, public health and preventive medicine [community medicine], otolaryngology, radiation oncology) with other fields (e.g., orthopedic surgery) expected to soon have similar challenges . Students may vote with their feet, with fewer applying for training in these fields . Students are asking for better career planning tools . A motion passed at the 2011 cma annual meeting called upon the organization to provide job - trend data and other career planning resources . Another resolution will compel the association to promote a national alignment of training positions with current and future societal needs, evolving models of care delivery and available health system resources . More effective marketing of geriatrics to both medical students and postgraduate trainees might bear fruit . More re - entry training positions for cfpc and rcpsc - accredited training would allow greater opportunities for practicing physicians to re - tool as specialists in geriatrics . This would require funding and the allocation of residency positions . A number of the functional specialists listed in table 2 were trained on the job . Allowing more international medical graduates with geriatric training to enter the country is a possible short - term response to our current shortfall . We would need to ensure that the training and skills they bring are congruent with our national needs, they remain in geriatrics after moving to canada, and their recruitment does nt disadvantage canadian trainees there are ethical concerns about recruiting internationally - trained health professionals from developing countries that also require consideration . Having trained individuals leave geriatrics is a regrettable and potentially preventable loss of physician resources . Retention appears to be a particular issue for those with care of the elderly training . A survey of care of the elderly graduates indicated that over half (55.8%) end up in a general family medicine practice . Since the development of this postgraduate training option in 1989, an estimated 160170 physicians (the cfpc does nt have a precise cumulative count) have completed training . Based on our determination of functional specialists, an estimated 40 of these physicians is examining the time spent in other clinical areas by practicing geriatricians . Among rcpsc - accredited specialists we need to assess the relative benefits and negative consequences of this on specialized geriatric services in our country . For example, specialists in geriatric medicine often attend on medical teaching units and/or work as internists in emergency rooms . In addition to providing a greater opportunity for them to instruct medical students and postgraduate trainees on the principles of geriatric care, there could be direct benefits for the many older patients they would see . Because of the unique knowledge, skill set, and approach they bring to the care of these patients, their involvement might lead to better outcomes such as lower mortality rates, better functional outcomes, less iatrogenesis, a greater proportion discharged back home, and/or lower readmission rates (these assertions could be evaluated in a pragmatic clinical trial). We have to weigh, though, the opportunity cost (i.e., the benefit that could have arisen by taking alternative action) of the time spent in non - geriatric activities and the risk of spreading ourselves too thin, which could have negative impacts on both the quality of work provided and its long - term sustainability . With our limited physician resources, we have to carefully prioritize the various ways we allocate our time . Concurrently we should point out to decision - makers the additional meritorious activities we could perform if we had sufficient numbers . According to the cma masterfile, 82 of the 233 (35.2%) certified specialists in geriatrics where age is known are 55 years of age and older . While hard to precisely predict when a given physician will retire, many practicing specialist physicians are closer to the end of their careers than to the beginning . If one assumes that physicians retire 40 years after receiving their md degree, we can estimate the expected number of retirements over the coming years . We should not focus solely on the complete cessation of work as prior to this, many physicians will reduce their workload and/or narrow their scope of practice, which has implications for physician work force planning . Strategies to keep older physicians engaged would include creating more job opportunities in non - clinical areas and flexibility in structuring part - time work . This is critical in all supply - side attempts to deal with a deficient number of practitioners . While efforts to improve physician recruitment and retention have historically focused on financial incentives, other ones should be considered . The overlapping factors that influence career decisions can be categorized as educational (e.g., early and on - going exposure to the field, length and availability of training opportunities, exposure to role models / mentors), professional (e.g., availability of positions, scope and type of practice, workload, frequency and intensity of on call responsibilities, professional support and collegiality, administrative support and availability of infrastructure, opportunities for professional development), personal or lifestyle (e.g., pre - existing interests and life experiences, desire for work - life balance, opportunities for part - time work / job sharing, family - friendly work environment, quality of life), and financial (e.g., level and stability of income, reimbursement for overhead expenses, availability of bursaries, debt relief / assistance, student loan forgiveness). A recent report by the institute for clinical evaluative sciences showed that mean and median payments to geriatricians remained significantly below the average for all physicians in ontario . (note: at the time this report was written, similar data were not available for other provinces .) Poor remuneration has long been perceived by many geriatricians as a major barrier to recruitment . While physicians should not be bribed to work in geriatrics, remuneration should not be a disincentive to considering the field . To make serious inroads on these issues will require a long - term commitment on the part of practitioners, medical and health leaders, and the cgs . Our ultimate goal is to improve the accessibility, effectiveness, and efficiency of specialized geriatric services . The first goal in particular might benefit from the innovative use of technology (e.g., telehealth, decision - support information technology) and labour substitution (e.g., expanding scopes of practice for other health professionals). Specialist physicians in geriatrics require infrastructure (e.g., interdisciplinary teams) to be fully effective . It is important to ensure that these teams are funded and available to them . Over time, new approaches and alternative models of care for older patients will be developed . As a specialty we should support their creation, critical evaluation, and dissemination, if found to be worthwhile . We view this report as just a start with further work required, especially as we move into areas such as assessing need and projecting requirements into the future . As noted, the values presented (especially for ftes) are imprecise because of the general definition we used for specialist physicians in geriatrics, the lack of a clear description of a fte in geriatrics, the need to rely on the personal knowledge of provincial / territorial leads about local physician resources and/or the collection of primary data, difficulties obtaining confirmatory information, and limited resources and time . More precise data would require clearer definitions for both a specialist and an fte, consistently adhering to these definitions in determining ftes, and systematically obtaining primary information . This would be an opportune time for the cgs to become more involved in physician resource planning . In addition to this being critical for the future health of this field of practice, there is increasing interest in aligning specialty training with societal needs . Health human resource planning aims at having the right number and type of practitioners available to provide the right services to the right people at the right time . In an efficient system, there would be timely implementation of effective policies to address discrepancies between requirement and supply . Canada has lagged behind other developed countries in creating a national strategy for planning the future supply of practitioners . Deals with health human resource planning: in the context of an evolving health - care system, the pgme [postgraduate medical education] system must continuously adjust its training programs to produce the right mix, distribution, and number of generalist and specialist physicians including clinician scientists, educators, and leaders to serve and be accountable to the canadian population . One issue the cgs should consider is whether specialists in geriatrics can and/or should describe themselves as generalists (for older people). The preceding report calls for more generalists which from their perspective includes general internists, general surgeons, general psychiatrists, and general pediatricians as well as family physicians . We found that the total number of specialist physicians is greater than the current membership of the cgs . We encourage the society to reach out to those who are not members and invite them to join . The society will have to reassure these potential members that it speaks to and for their interests . It is important for the society to start a dialogue with the canadian academy of geriatric psychiatry, long - term care medical directors association of canada, and others on the supply side of the equation . Bridges have to be built with potential allies like the alzheimer society of canada, who in their report on the future impact of the rising burden of dementia on canadian society advocated for more geriatricians . The society can play an important role by providing accurate information, informed advice, and perspective on physician resource issues . For example, in an otherwise sympathetic commentary about limited access to geriatricians, a report by the canadian institute for health information stated that the situation was improving because the number of medical doctors entering geriatrics training increased by 27% between 2005 and 2010 . This reassuring statement was based on an absolute increase of 4 in the total number of geriatric medicine trainees (from 15 in 20052006 to 19 in 20102011). Comparing 20082009 to 20102011 could have led to the depressing conclusion that the number had decreased 24% (from 25 to 19). We feel this is an example of mistaking noise (background irrelevant data) to signal (useful information). Wg recommendations submitted to the council of the cgs were: the over - riding interest of the cgs in physician resource planning must be on ensuring that older canadians have access to an effective, efficient, and humane health - care system . This will entail being clear on what is truly important, open - minded and flexible on non - critical issues, and willing to balance the ideal with the feasible.if the cgs wishes to be a credible voice in the field of physician work force planning, it must provide a balanced, reasoned perspective and allocate funding (and other resources) to the activity . While without doubt we face challenges, being unreasonably gloomy and/or making unrealistic projections about future need will potentially drive away recruits, reinforce the belief that it is already too late, and/or contribute to the sense that geriatrics is a luxury item.future specialist physician resource planning in geriatrics should focus on determining the ftes of functional specialists required . In 2006, the national advisory council on aging stated that we needed better data on the number of fte specialists in geriatrics.while we are confident that the current ftes of specialist physicians in geriatrics is less than the number required, at this time we can give no precise estimate of the present and future need for them . The cgs should support the determination of current and future physician resource needs using appropriate evidence - based methodologies.physician resource planning not linked to effective action will be futile . A national action plan should be multi - faceted, long - term, and modifiable as circumstances change . All should be explored and wherever appropriate and feasible supported by the cgs.the education of physicians without advanced training in geriatrics about the appropriate care of older patients is an essential component of the response of the health - care system to societal aging . While the cfpc standards for accreditation of family medicine training programs have specific requirements dealing with education about the care of older patients, they are generally lacking in rcpsc - accredited specialty and subspecialty programs . It is currently possible in canada to complete eight or more years of undergraduate and postgraduate medical training and receive a total of 10 hours of mandatory teaching in geriatrics.given their crucial role in undergraduate and postgraduate training of physicians, the cgs must engage the association of faculties of medicine of canada, cfpc, rcpsc, and collge des mdecins du qubec in addressing the issues raised in this report . The over - riding interest of the cgs in physician resource planning must be on ensuring that older canadians have access to an effective, efficient, and humane health - care system . This will entail being clear on what is truly important, open - minded and flexible on non - critical issues, and willing to balance the ideal with the feasible . If the cgs wishes to be a credible voice in the field of physician work force planning, it must provide a balanced, reasoned perspective and allocate funding (and other resources) to the activity . While without doubt we face challenges, being unreasonably gloomy and/or making unrealistic projections about future need will potentially drive away recruits, reinforce the belief that it is already too late, and/or contribute to the sense that geriatrics is a luxury item . Future specialist physician resource planning in geriatrics should focus on determining the ftes of functional specialists required . In 2006, the national advisory council on aging stated that we needed better data on the number of fte specialists in geriatrics . While we are confident that the current ftes of specialist physicians in geriatrics is less than the number required, at this time we can give no precise estimate of the present and future need for them . The cgs should support the determination of current and future physician resource needs using appropriate evidence - based methodologies . . A national action plan should be multi - faceted, long - term, and modifiable as circumstances change . The education of physicians without advanced training in geriatrics about the appropriate care of older patients is an essential component of the response of the health - care system to societal aging . While the cfpc standards for accreditation of family medicine training programs have specific requirements dealing with education about the care of older patients, they are generally lacking in rcpsc - accredited specialty and subspecialty programs . It is currently possible in canada to complete eight or more years of undergraduate and postgraduate medical training and receive a total of 10 hours of mandatory teaching in geriatrics . Given their crucial role in undergraduate and postgraduate training of physicians, the cgs must engage the association of faculties of medicine of canada, cfpc, rcpsc, and collge des mdecins du qubec in addressing the issues raised in this report.
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A 75-year - old male farmer presented to a primary care skin cancer practice in west australia with a 6-month history of an enlarging painless lesion on his left cheek with no history of any possible precursor lesion . There was a past history of treatment of two separate squamous cell carcinomas in - situ, one on the forehead and the other on an ear, in the previous two years . Other comorbidities included asthma, coronary heart disease, chronic obstructive pulmonary disease and arthritis . There was no history of nevus sebaceous and there was neither history nor clinical signs suggestive of cowden s syndrome . Examination showed a single small non - pigmented nodular lesion located on the left cheek in continuation with a small, pigmented macular lesion (figure 1). Dermatoscopy (figure 2) revealed a raised pink lesion, 3.5 mm in diameter, with a radial arrangement of vessels peripherally, with centered blood vessels in the middle of the lesion, these vessels being centered in skin - colored clods . No keratin structures (keratin scale, white structureless areas or white circles) were seen . At one edge of this lesion, in direct contact with it, was a heavily pigmented flat lesion, 1 mm in diameter with a pattern of pigmented lines radial, converging (figure 2). The lesion was subjected to an excision biopsy with a 4 mm peripheral margin and submitted for dermatopathological evaluation . Dermatopathology (figure 3) showed both the trichilemmoma and bcc (basal cell carcinoma) components . The trichilemmoma (figure 3 a1 and a2) was a well - circumscribed, sharply demarcated lesion with surface papillomatosis, horn cysts and a degree of central desmoplasia . Under high power an eosinophilic mantle of thickened basement membrane, mimicking the outer root sheath of the hair follicle infundibulum, was seen . Peripheral palisading was evident adjacent to the basement membrane and extending from that there were clear cells which were banal cytologically, lacked mitosis or individual cell necrosis (apoptosis) and contained an abundant amount of glycogen within their cytoplasm . The bcc component (figure 3 b1 and b2) showed superficial bcc at the dermo - epidermal junction . In the high power view melanin pigment trichilemmoma is a benign tumor derived from the external sheath cells of pilosebaceous units and desmoplastic trichilemmoma is a rare variant, which can present clinically with features suggesting invasive malignancy . There has been controversy as to whether trichilemmomas are actually aged warts, this belief being proposed by well - known dermatopathologist bernie ackerman but vigorously opposed by others including marty brownstein . Basal cell carcinoma is a malignant tumor, of trichoblast differentiation but uncertain origin, although it is probably of follicular derivation, predilection for non - glabrous skin being consistent with this . Trichilemmoma has previously been reported in association with bcc in the context of lesions arising in a preexisting nevus sebaceous . In addition, three cases of desmoplastic trichilemmoma arising in association with bcc without a preexisting nevus sebaceous have been reported . The dermatoscopy of four trichilemmomas has been reported with images being presented for three of them . In the first case keratin masses; perivascular whitish halos . The other three were reported together, with the first two described as having a hyperkeratotic central area with peripheral erythematous radial lines, the third having a peripheral radiated red area . All three were then presented as depicting what the authors called a red iris like structure . Examination of the photographs published in fact displayed a non - pigmented lesion in each case with a radial arrangement of vessels peripherally and structureless white centrally with some centred vessels . In the case we report, the clinician interpreted the lesion as a pigmented lesion and assessed it using the chaos and clues decision - algorithm . The lesion was regarded as chaotic (asymmetrical) with the clues of an eccentric structureless (pink) area as well as lines radial segmental, which lead to a clear decision to do an excision biopsy . The pathologist, as is often the case with esoteric lesions, was able to deliver the answer to which particular type of malignancy this was . The dermatoscopic features of the trichilemmoma presented here are consistent with those published previously, with a radial pattern of vessels peripherally and centred vessels centrally . Association with a bcc is a previously reported, unusual variation, consistent with both lesions having hair - follicle derivation . This case demonstrates the utility of using a decision algorithm in esoteric cases with the final diagnosis to be delivered by the gold standard of dermatopathology.
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A total of 146 consecutive patients (292 lower limbs), referred to the physiology department for doppler ultrasound evaluation of pad, were prospectively included in the study . Risk factors were self reported by the patient, and biological values were found in medical files . The lower limbs were classified as clinically normal when skin was normal and both dorsalis pedis and posterior tibialis pulses were present . Doppler measurement and two - dimensional ultrasound examination were conducted by a single investigator (c.c . ), using the toshiba powervision 7000 10-mhz linear probe . Two - dimensional images and doppler interrogation were obtained for iliac to ankle arteries and the abdominal aorta . Stenoses were evaluated with the ratio of the maximal systolic velocity at stenosis to the systolic velocity proximal to the stenosis . Osc - abi was measured with an automatic device (dynamap 8100, critikon, little chalfont, buckinghamshire, u.k .) By nurses or clinical staff . Systolic blood pressure was measured with the cuff placed above the ankles for both legs and both arms . Nurses or clinical staff were taught to measure osc - abi, i.e., to install the cuff with the sampling area facing the ankle artery, namely posterior, and anterior tibial artery for ankle measurement and brachial artery . Dop - abi was measured by a single investigator (c.c .) With a continuous wave doppler device (md2, 8-mhz probe, huntleigh, luton, u.k . ). Abi was computed as the ratio of ankle blood pressure to the highest brachial systolic blood pressure for both osc - abi and dop - abi . Costs of procedures were derived from the french medical care system fees for doppler ultrasound examination or abi measurement, combined with a visit with a cardiologist, a general practitioner, or a nurse . Qualitative and quantitative variables were compared using and student's t tests, respectively . The level of agreement between osc - abi and dop - abi was assessed both by correlation analysis using pearson's correlation coefficient and by a bland - altman plot (13). Receiver operating characteristic (roc) curves were computed as well as areas under roc curves using proc logistic; curve areas were compared using a nonparametric approach (14) implemented in an sas macro for correlated data or a wald test (15). Positive predictive value (ppv) and negative predictive value (npv) were computed . The positive likelihood ratio was computed as the probability of true positive over the probability of false positive, and the negative likelihood ratio was computed as the probability of false negative over the probability of true negative . Doppler measurement and two - dimensional ultrasound examination were conducted by a single investigator (c.c . ), using the toshiba powervision 7000 10-mhz linear probe . Two - dimensional images and doppler interrogation were obtained for iliac to ankle arteries and the abdominal aorta . Stenoses were evaluated with the ratio of the maximal systolic velocity at stenosis to the systolic velocity proximal to the stenosis . Osc - abi was measured with an automatic device (dynamap 8100, critikon, little chalfont, buckinghamshire, u.k .) By nurses or clinical staff . Systolic blood pressure was measured with the cuff placed above the ankles for both legs and both arms . Nurses or clinical staff were taught to measure osc - abi, i.e., to install the cuff with the sampling area facing the ankle artery, namely posterior, and anterior tibial artery for ankle measurement and brachial artery . Dop - abi was measured by a single investigator (c.c .) With a continuous wave doppler device (md2, 8-mhz probe, huntleigh, luton, u.k . ). Abi was computed as the ratio of ankle blood pressure to the highest brachial systolic blood pressure for both osc - abi and dop - abi . Costs of procedures were derived from the french medical care system fees for doppler ultrasound examination or abi measurement, combined with a visit with a cardiologist, a general practitioner, or a nurse . Qualitative and quantitative variables were compared using and student's t tests, respectively . The level of agreement between osc - abi and dop - abi was assessed both by correlation analysis using pearson's correlation coefficient and by a bland - altman plot (13). Receiver operating characteristic (roc) curves were computed as well as areas under roc curves using proc logistic; curve areas were compared using a nonparametric approach (14) implemented in an sas macro for correlated data or a wald test (15). Positive predictive value (ppv) and negative predictive value (npv) were computed . The positive likelihood ratio was computed as the probability of true positive over the probability of false positive, and the negative likelihood ratio was computed as the probability of false negative over the probability of true negative . Diabetic patients had microvascular disease with nephropathy (34.6%), retinopathy (35.8%), and neuropathy (48.1%) or macrovascular disease (40.7%) including coronary artery disease, pad, stroke, or carotid stenosis . Pad was clinically suspected because of the absence of distal artery pulses in 54% of nondiabetic patients and 40% of diabetic patients . Pad was diagnosed with dop - abi in 33% of nondiabetic patients and 27% of diabetic patients . Doppler ultrasound examination showed at least one arterial stenosis in 41.7% of the legs in diabetic patients and in 50.8% of the legs in nondiabetic patients . A proximal localization (i.e., iliac or femoral artery) was more frequent in nondiabetic than in diabetic patients (37.7 and 5.2% of the localizations, respectively, p <0.0001), who had more distal localization (72.0 vs. 20.8% of the localizations in nondiabetic patients, p = 0.0004). Population characteristics data are means sd (range) or n (%). * p <0.05 with t test comparison of means or test comparison of frequencies . Dop - abi was measurable in 98% of the total population studied (97% of diabetic and 99.2% of nondiabetic patients), whereas osc - abi was obtained in 95.5% of the legs in the whole population (p = 0.16 for comparison of techniques), in 95.8% of diabetic patients, and in 95.3% of nondiabetic patients . When osc - abi was not measurable, dop - abi was either not measurable or <0.90 in most patients (9 of 13 legs). Osc - abi was related to dop - abi (r = 0.53, p <0.0001) in the total population, as well as in nondiabetic patients and in diabetic patients (fig . Bland - altman representation showed that dop - abi and osc - abi were in good agreement in a wide range of values, and the difference did not vary with the average values in any significant way (fig . The mean difference between the two methods was 0.0207 0.27 in the total population, 0.028 0.25 in diabetic patients, and 0.0098 0.28 in nondiabetic patients . Relationship between dop - abi and osc - abi in nondiabetic (a) (r = 0.60; p <0.0001) and diabetic (b) (r = 0.49; p <0.0001) patients . C: bland - altman representation of doppler and oscillometric measurement of abi in the whole population and the 95% limit of agreement to the mean difference . Roc curves constructed for osc - abi with dop - abi 0.90 defining pad (fig . Areas under the curve (aucs) of roc curves were not different in diabetic and nondiabetic patients (auc 0.83 and 0.844, respectively, p = 0.66). The best cutoff value for optimal sensitivity and specificity was 1.02 in the whole population, 1.04 in diabetic patients, and 1.0 in nondiabetic patients (fig . Roc curves for osc - abi for diagnosis of pad, as defined with dop - abi <0.90 in the whole population (a), diabetic patients (b), and nondiabetic patients (c). D: best cutoff determination for maximizing both the sensitivity and the specificity in the whole population, diabetic and nondiabetic patients ., roc curves were constructed for dop - abi and osc - abi with stenosis at ultrasound as the criterion for diagnosis of pad (fig . The roc curve analyses showed that dop - abi was better than osc - abi for correctly classifying patients for pad (aucdop - abi 0.873, aucosc - abi 0.806, p = 0.026). Dop - abi performance was equivalent in nondiabetic and diabetic patients (p = 0.068 for aucs of roc curves), whereas osc - abi did not perform as well as dop - abi in diabetic patients (p = 0.026 for roc auc comparison). Roc curves for dop - abi and osc - abi for diagnosis of ultrasound - identified arterial stenosis in the whole population (a), diabetic patients (b), and nondiabetic patients (c). E: dop - abi best cutoff determination for maximizing both the sensitivity and the specificity in the whole population, diabetic and nondiabetic patients ., sensitivity;, specificity . The diagnostic accuracy for selected abi thresholds (table 2) showed that the commonly used value of 0.90 has a high specificity but a medium sensitivity in the whole population . The sensitivity of dop - abi and osc - abi was greater in the nondiabetic population compared with that in diabetic patients . Thus, a threshold value of 0.90 defined a highly specific test at the expense of sensitivity for both dop - abi and osc - abi . Dop - abi and osc - abi presented high ppvs in the total population (95.3 and 88.1%, respectively) but also in diabetic patients (92.5 and 83.3%, respectively). Conversely, npvs were medium in both the whole population (76.1 and 65.9%, respectively) and diabetic patients (74.7 and 66.2%, respectively). To optimize the threshold values of abi for screening and diagnosis, respectively, figure 3b and c shows that the highest sensitivity and specificity were achieved at cutoff values between 1 and 1.1 for both dop - abi and osc - abi in patients with or without diabetes . Diagnostic performance of dop - abi and osc - abi for diagnosis of ultrasound - identified arterial stenosis in the total population and in nondiabetic and diabetic patients nlr, negative likelihood ratio; plr, positive likelihood ratio . According to the french medical care system, the costs of pad screening would be 121 (162 us dollars [usd]) with a vascular ultrasound study only or 66 (88 usd) with abi only by a cardiologist, 43 (58 usd) with abi only by a general practitioner, and 33 (44 usd) with abi only by a nurse . Dop - abi was measurable in 98% of the total population studied (97% of diabetic and 99.2% of nondiabetic patients), whereas osc - abi was obtained in 95.5% of the legs in the whole population (p = 0.16 for comparison of techniques), in 95.8% of diabetic patients, and in 95.3% of nondiabetic patients . When osc - abi was not measurable, dop - abi was either not measurable or <0.90 in most patients (9 of 13 legs). Osc - abi was related to dop - abi (r = 0.53, p <0.0001) in the total population, as well as in nondiabetic patients and in diabetic patients (fig . Bland - altman representation showed that dop - abi and osc - abi were in good agreement in a wide range of values, and the difference did not vary with the average values in any significant way (fig . The mean difference between the two methods was 0.0207 0.27 in the total population, 0.028 0.25 in diabetic patients, and 0.0098 0.28 in nondiabetic patients . Relationship between dop - abi and osc - abi in nondiabetic (a) (r = 0.60; p <0.0001) and diabetic (b) (r = 0.49; p <0.0001) patients . C: bland - altman representation of doppler and oscillometric measurement of abi in the whole population and the 95% limit of agreement to the mean difference . Roc curves constructed for osc - abi with dop - abi 0.90 defining pad (fig . Areas under the curve (aucs) of roc curves were not different in diabetic and nondiabetic patients (auc 0.83 and 0.844, respectively, p = 0.66). The best cutoff value for optimal sensitivity and specificity was 1.02 in the whole population, 1.04 in diabetic patients, and 1.0 in nondiabetic patients (fig . Roc curves for osc - abi for diagnosis of pad, as defined with dop - abi <0.90 in the whole population (a), diabetic patients (b), and nondiabetic patients (c). D: best cutoff determination for maximizing both the sensitivity and the specificity in the whole population, diabetic and nondiabetic patients ., roc curves were constructed for dop - abi and osc - abi with stenosis at ultrasound as the criterion for diagnosis of pad (fig . The roc curve analyses showed that dop - abi was better than osc - abi for correctly classifying patients for pad (aucdop - abi 0.873, aucosc - abi 0.806, p = 0.026). Dop - abi performance was equivalent in nondiabetic and diabetic patients (p = 0.068 for aucs of roc curves), whereas osc - abi did not perform as well as dop - abi in diabetic patients (p = 0.026 for roc auc comparison). Roc curves for dop - abi and osc - abi for diagnosis of ultrasound - identified arterial stenosis in the whole population (a), diabetic patients (b), and nondiabetic patients (c). E: dop - abi best cutoff determination for maximizing both the sensitivity and the specificity in the whole population, diabetic and nondiabetic patients . The diagnostic accuracy for selected abi thresholds (table 2) showed that the commonly used value of 0.90 has a high specificity but a medium sensitivity in the whole population . The sensitivity of dop - abi and osc - abi was greater in the nondiabetic population compared with that in diabetic patients . Thus, a threshold value of 0.90 defined a highly specific test at the expense of sensitivity for both dop - abi and osc - abi . Dop - abi and osc - abi presented high ppvs in the total population (95.3 and 88.1%, respectively) but also in diabetic patients (92.5 and 83.3%, respectively). Conversely, npvs were medium in both the whole population (76.1 and 65.9%, respectively) and diabetic patients (74.7 and 66.2%, respectively). To optimize the threshold values of abi for screening and diagnosis, respectively, figure 3b and c shows that the highest sensitivity and specificity were achieved at cutoff values between 1 and 1.1 for both dop - abi and osc - abi in patients with or without diabetes . Diagnostic performance of dop - abi and osc - abi for diagnosis of ultrasound - identified arterial stenosis in the total population and in nondiabetic and diabetic patients nlr, negative likelihood ratio; plr, positive likelihood ratio . According to the french medical care system, the costs of pad screening would be 121 (162 us dollars [usd]) with a vascular ultrasound study only or 66 (88 usd) with abi only by a cardiologist, 43 (58 usd) with abi only by a general practitioner, and 33 (44 usd) with abi only by a nurse . Automated oscillometric abi measurement by clinical assistants with little formal training was shown to be feasible in most patients, including diabetic patients . The diagnostic efficiency was close to that of dop - abi in nondiabetic as well as in diabetic patients, as validated against ultrasound . Furthermore, we defined threshold values for osc - abi measurement as a screening or diagnostic test for pad in nondiabetic and diabetic patients . Our study showed that dop - abi and osc - abi were significantly related, as previously reported in several studies (11,12,16). Both methods were limited in some patients (0.6% of the legs in our population) when lower limb arteries were not compressible . We also described, for the first time, evaluation of the use of the oscillometric method in diabetic patients . Indeed, we showed that osc - abi was obtained in most diabetic patients and related to dop - abi . Furthermore, the low rate of failure for osc - abi (1.3% of diabetic patients) showed that most arteries could be evaluated with this method . The availability of an automated oscillometric method in diabetic subjects is of particular interest because pad is frequently associated with few clinical symptoms (10). Indeed, we validated both dop - abi and osc - abi against ultrasound as a reference and osc - abi against dop - abi as a reference in diabetic patients and established threshold values for detection of artery stenosis . The analysis of roc curves showed that dop - abi was slightly better than osc - abi in diabetic patients . Despite this difference, osc - abi could represent an alternative method for abi measurement, even in diabetic patients, with the benefit that this method is more readily available than doppler measurement . Our study further extended the evaluation of dop - abi and osc - abi as a diagnostic or a screening test . Setting the threshold at 0.90 led to a ppv> 95% and a threshold of 1.1 led to an npv> 99% (1719). In our study, dop - abi showed a similar ppv (95%) at a threshold of 0.90 but a lower npv (76%) with a threshold of 1.1 . This result may be related to differences in the populations studied: the relatively smaller size of our population and higher - risk patients . We demonstrated similar results with osc - abi and dop - abi in the same patients, with ppv and npv that were 10% lower in the diabetic subpopulation . Despite this difference, the diagnostic accuracy of osc - abi remained high enough to conclude that it could help in pad assessment in diabetic patients . Indeed, pad is frequent in diabetic patients, despite missing or confounding clinical symptoms, because of its coexistence with neuropathy (10). Thus, the use of osc - abi as a routine test could improve pad diagnosis and management of lower limb complications in diabetes (8). Further, we established threshold values of abi optimized for a screening purpose . The roc curves showed that both dop - abi and osc - abi could be used for screening for arterial stenosis with a cutoff set at 1.0 to 1.1, which identified> 80% of the patients with pad whatever the technique used and the presence or absence of diabetes . These results were in agreement with those reported for dop - abi with a normal value between 1 and 1.2 (2,5,17,20). Obstacles to adopting abi measurement for pad diagnosis in primary care were identified previously as time, cost, and the need for a dedicated device and operator skill (6,7,12). In contrast, we have demonstrated that osc - abi can be easily performed by clinical assistants, i.e., nurses or others, after minimal training and with commonly available devices (2,7). This approach represents substantial savings of costs and time, with a significant improvement in the diagnosis and management of pad in the general population and particularly in diabetic patients . Our study was not designed for cost - effectiveness evaluation; however, we showed that the use of osc - abi, as first - line screening for pad, would reduce by 61 and 75% the use of vascular studies for pad diagnosis (25 of 63 nondiabetic patients and 21 of 83 diabetic patients). In patients in whom distal artery pulses were not palpable, vascular studies would have been reduced by 24 and 36% in nondiabetic and diabetic patients, respectively . The reduction of costs (roughly divided by a factor of 3) clearly supports the hypothesis of cost saving in pad diagnosis with the use of osc - abi, as measured by nurses and clinical staff . Moreover, the early recognition of pad would improve the management of lower limb complications and cardiovascular risk factors in the general population, with a further reduction in costs because of early intervention for modifiable cardiovascular risk factors (10,21). Nevertheless, the cost - effectiveness of osc - abi needs to be assessed in a study specifically design for that purpose . In summary, our study showed that osc - abi measurement was feasible for clinical assistants using an automatic blood pressure monitoring device . This may be a low - cost and effective procedure to improve pad management in at - risk patients, including subjects with diabetes, with early recognition and appropriate counseling for the control of modifiable cardiovascular risk factors.
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Acute poisonings with caustic substances can cause severe chemical injuries to the upper gastrointestinal tract, which can be localized from the mouth to the small intestines . They are a result of a sudden or intentional ingestion of caustic substances and can be seen in patients of different ages . These kind of poisonings often end up with post corrosive complications, such as stenosis, but they can also have a fatal ending due to the perforation of the gastrointestinal lumen or tracheal necrosis (1 - 3). Severity of the post corrosive chemical injuries of the upper gastrointestinal tract depends on nature of the caustic substance, its quantity and concentration, time of exposure, act of swallowing and many other factors(4). Unlikely in children, where caustic poisonings are most often accidental, in adult patients these poisonings are intentional, with suicidal attempts (> 90%)(5). In many countries there are educational programs and activities, organized among population with lower health culture, all with an aim to explain the danger of abuse of caustic chemicals and consequences of ingestion . Regardless, the number of caustic poisonings is very high, they have severe clinical expression, clinical diagnostic investigations are hard to perform, and outcome is often uncertain (6). The annual report of the american poison control center from 2011 reports that from the total number of registered poisonings 202056 are intoxications due to abuse of cleaning agents, which are in the group of chemical agents with mainly corrosive characteristics . A five year study conducted in turkey stated that 2.5% of the total numbers of poisonings are caustic poisonings . A similar study performed in england and wales showed that a third of the total number of poisonings are due to the abuse of agents used for maintaining of the hygiene in the households, and these poisonings are more often among the adults, alike as among children (7 - 9). Many authors consider that findings from the urgent esophagogastroduodenoscopy can be used as a basis for treatment of caustic poisonings . They are many endoscopic classifications of the post corrosive injuries, but we use the one suggested by kikendal . The treatment is strictly surgical in patients with severe and life - threatening complications (10 - 11). Through literature there are many reports that show the mortality rate in acute caustic poisonings and all the effort that should be made to reduce it . The aim of this study is to examine the influence of patient s age on the mortality rate and survival of patients with acute caustic poisonings, and also to analyze their correlation . We studied medical records from 415 patients, aged between 14 and 90 years, who were hospitalized and treated, in the period between 2007 and 2011 . After admission to hospital all patients were immediately situated at the intensive care unit (icu) and in the first 12 - 24 hours all of them went under urgent esophagogastroduodenoscopy . It was performed using an endoscope for upper endoscopy, type olympus (japan) with a diameter of 9.2 mm . Control esophagogastroduodenoscopy was carried out after 15 to 25 days after caustic ingestion . For a better accomplishment of the procedure we prepared the patients with pre - medication with xylocaine gel, as a local anesthetic . Methods as insufflations and retro - visualization were performed very carefully because of the great risk from causing additional iatrogenic injuries . Post - caustic injuries are classified according to the kikendal s classification (table 1). Clinical characteristics of patients patients, according to their age, were divided in three groups: group of patients aged between 14 and 35 yearsgroup of patients aged between 35 and 55 years andgroup of patients older than 55 years . Group of patients aged between 14 and 35 years group of patients aged between 35 and 55 years and group of patients older than 55 years . They were also divided according to their gender, purpose of intoxication, type of abused corrosive substance and severity of post - corrosive injuries . We analyzed the influence of age on several parameters: need of intensive care, duration of stay in the intensive care unit, duration of hospitalization, severity of post - corrosive lesions on the upper git, mortality and survival rates . In the survey we included 415 patients with acute corrosive poisonings, from which 295 (71.08%) were females and 120 (28.92%) were males . 388 (93.49%) from the total number of patients ingested the corrosive agent with suicidal attempt and 27 (6.5%) ingested it accidentally . The age of patients varieties in the interval 41.3818.06 years, cl: 39, 64 - 43, 13; where the minimal age is 14 years, and maximal 90 years . Distribution of patients age in different ages groups showed that the majority of patients 181 (43.61%) belong in the first group, subjects aged between 14 and 35 years, 144 (34.70%) were patients aged between 35 and 55 years, and only 90 (21.69%) were patients older than 55 years . Greater number of patients, 388 (93, 49%), ingested the corrosive agent on purpose and the majority of them abused hydrochloric acid . According to the classification of kikendal the greater number of patients had post - corrosive lesions of grade ii a (table 2). Degree of post - corrosive damages and mortality rates results regarding on differences in duration of stay in icu, also considering the distributions of patients in different ages groups, showed that f=22.86 and p<0.001 (p=0.000) and there is a significant difference in the duration of stay along different ages groups in patients with corrosive poisonings . On graph 1 we show the relation between age of patient and stay in the icu . For r=0.32 (p<0.05) in fact with greater age of patients the duration of stay in icu becomes longer and that is why the correlation between patients age and hospitalization in icu is significant . Figure 1 illustrates results from the difference in duration of hospitalization, considering distribution of patients in different age groups, which showed that for f=27.32 and p<0.001 (p=0.000) there is a significant difference in days of hospitalization in icu between the age groups of patient with corrosive poisonings . Cumulative proportion surviving correlation between different ages of patient and duration of hospitalization with r=0.30 (p<0.05) there is mild, but still significant correlation . Studied correlation between ages of patients and grades of post - corrosive injuries showed strong and significant correlation for r=0.47 (p<0.05). The correlation between ages and grades of post - corrosive injuries for r=0.43 (p<0.05) showed mild, but significant correlation . We also analyzed the correlation between grades of post - corrosive lesions and mortality rate . For chi - square=316.49; df = 4 and p=0.000 * * * there is a significant correlation between mortality rate as dependent variable and influence of grades of post - corrosive injuries as independent variables (table 3). Descriptive analysis for age groups logistic regression analyses for mortality rate in acute corrosive poisonings as dependent variable and influence of age intervals as independent variables showed that for chi square=25.13 and p<0.001 (p=0.000) there is a significant correlation between mortality and influence of age . We took the group aged between 14 and 35 years as a referent category . From or values we can show that: patients older than 55 years have 11.06 times (or=11.06/exp(b)) greater risk of fatal ending of the poisoning correlated to patients aged between 14 and 35 years, in fact the influence of age higher than 55 years is significant (95.0% cl: 0.3, 62 - 33, 82/p<0.001(p=0.000)).patients aged between 35 and 55 years have 2.60 times (or=2.60/exp(b)) greater risk for fatal ending of the poisoning correlated to patients aged between 14 and 35 years, but the influence of this age group, 35 - 55 years, is not significant (95.0% cl:0, 77 - 8, 82/ p>0.05(p=0.125)).analyses of survival rates in 415 patients with caustic poisonings was in a follow up period of 180 days . Total 30 (7.23%) patients died from the intoxication and 385 (92.77%) patients (censored patients) were alive until the end of our study.patients aged between 14 and 35 years consisted of 177 (42.65%) patients and time of survival varied in the interval 176.0926.10 days.patients aged between 35 and 55 years consisted of 136 (32.77%) patients and the time of survival varied in the interval 170.0941.00 days.72 (16.87%) patients were older than 55 years and their survival time varied in the interval 144.9470.56 days.in the total number of patients (415) the survival time varied in the interval 167.2545.75 days (table 4).we can see that the majority of deaths were recorded in the first 10 days of follow-up.differences in survival between different age groupsfor chi=28.86 and p<0.001(p=0.000) there is a significant difference in the survival rates between different age groups.patients aged between 35 and 55 years have a shorter survival period compared to patients aged between 14 and 35 years, but the difference for log - rank test=1.58 and p>0.05 (p=0.11) is not significant . (log - rank test/14 - 35 years and 35 - 55 years / test statistic=-1.58; p=0.11).patients older than 55 years have shorter survival period compared to patients aged between 14 and 35 years and the difference in survival for log - rank test=-5.03 and p<0.001 (p=0.000) is significant . (log - rank test/14 - 35 years and> 55 years./test statistic=-5.03; p=0.000).patients older than 55 years have shorter period of survival compared to patients aged between 35 and 55 years and the difference in survival for log - rank test=-3.41 and p<0.01 (p=0.001) is significant . (log - rank test/35 - 55 years and> 55years./test statistic=-3.41;p=0.001).predictive role of different age groupsresults from cox s regression, the method used for calculating the predictive role of age groups on survival of patients with caustic poisonings, are shown on table 2.the patients aged between 14 and 35 years were taken as a referent category.patients aged between 35 and 55 years for hr=2.55; (95% cl for hr=0.77 - 8.45; p>0.05) have 2.55 times shorter survival time compared to referent group (14 - 35 years). The influence of age interval, 35 - 55 years, is not significant.patients older than 55 years for hr=9.65 (95%cl for hr=3.26 - 28.51;p<0.001) have 9.65 times shorter survival period than referent group (14 - 35 years). The influence of this age interval (> 55 years) is significant.statistical analysis of all provided date was made by using the statistical program statistica 7.1 and spss 13.0.in series with attributive characteristics we calculated structural percentages (%); differences in analyzed parameters in the series with attributive characteristics (gender, corrosive agents, age groups and systemic complications) were tested with pearson chi - square (x).we also provided results from descriptive statistics (mean std . Patients older than 55 years have 11.06 times (or=11.06/exp(b)) greater risk of fatal ending of the poisoning correlated to patients aged between 14 and 35 years, in fact the influence of age higher than 55 years is significant (95.0% cl: 0.3, 62 - 33, 82/p<0.001(p=0.000)). Patients aged between 35 and 55 years have 2.60 times (or=2.60/exp(b)) greater risk for fatal ending of the poisoning correlated to patients aged between 14 and 35 years, but the influence of this age group, 35 - 55 years, is not significant (95.0% cl:0, 77 - 8, 82/ p>0.05(p=0.125)). Analyses of survival rates in 415 patients with caustic poisonings was in a follow up period of 180 days . Total 30 (7.23%) patients died from the intoxication and 385 (92.77%) patients (censored patients) were alive until the end of our study . Patients aged between 14 and 35 years consisted of 177 (42.65%) patients and time of survival varied in the interval 176.0926.10 days . Patients aged between 35 and 55 years consisted of 136 (32.77%) patients and the time of survival varied in the interval 170.0941.00 days . 72 (16.87%) patients were older than 55 years and their survival time varied in the interval 144.9470.56 days . In the total number of patients (415) the survival time varied in the interval 167.2545.75 days (table 4). We can see that the majority of deaths were recorded in the first 10 days of follow - up . Differences in survival between different age groups for chi=28.86 and p<0.001(p=0.000) there is a significant difference in the survival rates between different age groups . Patients aged between 35 and 55 years have a shorter survival period compared to patients aged between 14 and 35 years, but the difference for log - rank test=1.58 and p>0.05 (p=0.11) is not significant . (log - rank test/14 - 35 years and 35 - 55 years / test statistic=-1.58; p=0.11). Patients older than 55 years have shorter survival period compared to patients aged between 14 and 35 years and the difference in survival for log - rank test=-5.03 and p<0.001 (p=0.000) is significant . (log - rank test/14 - 35 years and> 55 years./test statistic=-5.03; p=0.000). Patients older than 55 years have shorter period of survival compared to patients aged between 35 and 55 years and the difference in survival for log - rank test=-3.41 and p<0.01 (p=0.001) is significant . (log - rank test/35 - 55 years and> 55years./test statistic=-3.41;p=0.001). Predictive role of different age groups results from cox s regression, the method used for calculating the predictive role of age groups on survival of patients with caustic poisonings, are shown on table 2 . Patients aged between 35 and 55 years for hr=2.55; (95% cl for hr=0.77 - 8.45; p>0.05) have 2.55 times shorter survival time compared to referent group (14 - 35 years). Patients older than 55 years for hr=9.65 (95%cl for hr=3.26 - 28.51;p<0.001) have 9.65 times shorter survival period than referent group (14 - 35 years). Statistical analysis of all provided date was made by using the statistical program statistica 7.1 and spss 13.0 . In series with attributive characteristics we calculated structural percentages (%); differences in analyzed parameters in the series with attributive characteristics (gender, corrosive agents, age groups and systemic complications) were tested with pearson chi - square (x). We used kaplan - meier method, while differences in survival between two age groups were tested with log - rank test . Predictive value of influence of different age groups on survival period was calculated with cox regression method (hr with 95% cl). In series with numerical characteristics we calculated the following: differences between certain analyzed parameters (intensive care; hospitalization) in different age groups was tested using analysis of variance (f)/scheffe test;influence of age groups on duration of hospitalization in intensive care unit and duration of hospitalization in general, tested with multiple regression (r);prognostic value of age groups as independent variables on mortality rate, acute and late post - corrosive complications, tested with logistic regression (chi square, wald, exp(b));relation between age / age groups and grade of post - corrosive injuries, tested with spearman rank order correlations (r);relation between age of patients and stay in the intensive care unit, duration of hospitalization, tested with pearson coefficient for correlation (r). Differences between certain analyzed parameters (intensive care; hospitalization) in different age groups was tested using analysis of variance (f)/scheffe test; influence of age groups on duration of hospitalization in intensive care unit and duration of hospitalization in general, tested with multiple regression (r); prognostic value of age groups as independent variables on mortality rate, acute and late post - corrosive complications, tested with logistic regression (chi square, wald, exp(b)); relation between age / age groups and grade of post - corrosive injuries, tested with spearman rank order correlations (r); relation between age of patients and stay in the intensive care unit, duration of hospitalization, tested with pearson coefficient for correlation (r). Unregulated production, import, packing and labeling of various caustic agents, due to inappropriate legislative, made them one of the most often abused substances in everyday life, especially in developing countries where the number of caustic poisonings rises (12). Data for republic of macedonia state that caustic poisonings take 15 - 18% of the total number of poisonings, they are responsible for 14 - 27% of the complications and they have mortality rate of 4 - 6% (13). Intoxications with caustic substances cause severe post - corrosive complications in the upper part of the gastrointestinal tract, which can be influenced by age of the patients, gender, amount of ingested agent, duration of exposure and the time aframe between initiation of treatment and completion of urgent esophagogastroduodenscopy (14). Caustic substances cause tissue destruction due to coagulation or colliquative necrosis with high percentage of late post - corrosive complication of 15 - 85% . The mortality rate is still high despite the intensive hospital care, sophisticated diagnostic and therapeutically approaches . Among literature the role of therapy in acute caustic poisonings is to prevent perforation of the gastrointestinal wall and to avoid the processes as progressive fibrosis and stenosis of the esophagus and stomach . Studies state that elasticity of both esophageal and gastric walls, after caustic poisoning, is decreased due to the low collagen amount, which production is compromised . After progressive fibrosis many cases end up with narrowing of the gastrointestinal lumen, especially in the esophagus and stomach (16). Early and late post - corrosive complications in acute caustic poisonings are related with the severity of the post - caustic injuries, seen with upper endoscopy . They are also related with age of patients and time frame between intoxication and admission to hospital . Unwanted outcomes can be expected several hours or even several months or years after the poisoning (18). Age of patients, gender, time frame between ingestion and start of reanimation, consciousness, positive peritoneal signs, shock index, ph value and concentration of bicarbonates in the arterial blood can be used as prognostic factors for mortality in patients with acute caustic poisonings . Remarkable higher mortality rate is seen among patients who are 50 years, have positive peritoneal signs, shock index higher than 1, ph value of 7.2 or lower and bicarbonates concentration in arterial blood sample lower than 16 we confirmed that mortality rate and time of survival are significantly correlated with patients age . Another study also stated that number of older patients hospitalized at the intensive care unit is decreased and their cumulative survival rate was also lower compared to younger patients . However, despite the greater percentage of systematic complications, and also higher mortality rate in older patients (older than 65 years), seriousness of the post - corrosive injuries of the upper gastrointestinal tract did nt have great influence or did nt have any influence at all on the survival period . This fact is also shown in groups of younger patients (younger than 65 years) who had severe post - corrosive damages (grade iii b) (21). Acute caustic poisonings have great risk of fatal outcome especially during the acute phase (in the first 96 hours) and this is why we should pay special attention on quick transportation of the patients to specialized hospitals and their admission to the intensive care unit, where they should receive prompt therapy and also artificial ventilation if needed . Artificial nutrition should be administrated in the chronic phase of the poisoning, which can last up to few months (22 - 23). Estimation of cost / benefit in different therapeutically protocols and also duration of hospitalization are very complex and questionable topics . Doctors for a long time thought that cost of the treatment should not influence the decision making process related to the charge of the entire treatment and duration of hospitalization . Length of hospital stay and treatment in patients with acute caustic poisonings are of a great importance for the final outcome and also for the economical effect and reduction of the hospital costs . Many control studies conducted on large group of patients stated that hospitalization of these patients start in the intensive care units (icu), usually lasts 10 to 15 days, and can be postponed to 25 or 30 days depending on severity of the post - corrosive injuries of the upper gastrointestinal tract and occurrence of eventual late complications (24 - 25). Treatment of acute caustic poisonings should be accomplished in the intensive care unit (icu) due to the severe clinical condition of the majority of patients and also the need for intensive monitoring . This period usually lasts 10 to 20 days, actually until the processes of intensive granulation and healing of the caustic injuries are over . At the same time the patients require different types of artificial nutrition, which depends on the grade of the post - corrosive injuries . Hospital stay in these patients is significantly shorter after standardization of sophisticated ways of artificial nutrition (home enteral nutrition) (26 - 27). Post - corrosive complications may cause hemorrhages in the upper part of the gastrointestinal tract, even in the early acute phase, due to the damages of the esophageal and gastric walls, tracheal necrosis and esophageal or gastric perforation, which complications may cause serious and urgent state or even end up fatally . In this kind of cases the only treatment of choice is surgical, where the outcomes are always very unpredictable . Ingestion of greater amount of caustic substance with suicidal attempts usually causes injuries of the esophageal and gastric walls and they go through the muscular layer where the vascular net is very rich and its damage may cause serious and intensive bleeding or rupture of the wall and leakage of the gastrointestinal content in the mediastinal or peritoneal cavity . This can end up with mediatinitis or peritonitis, very urgent and dangerous complications which ask for urgent surgical interventions and are related to high mortality rates (28 - 30). Morbidity and mortality rates in acute caustic poisonings are still high and depend on the severity of the post - corrosive injuries of the upper gastrointestinal tracts, approved with urgent esophagogastroduodenscopy . Often these poisonings have unwanted outcomes even before the patient was administered to specialized hospital, where the treatment is very professional and sophisticated . Survival of these patients is still an imperative and it can be prolonged with prompt diagnosis, prevention of perforation and quick treatment with intensive hyper alimentation during the acute phase (first 15 days) and control of fatal infections . During the chronic phase we should pay special attention to surgical treatment and appropriate decision for treating late chronic complications surgically (stenosis of esophagus and stomach) (31). Mortality rates in acute caustic poisonings are still very high and are a serious problem during the treatment . Percentage of fatal outcomes is higher during the acute phase of treatment (first 96 hours), usually a result of gastrointestinal complications (perforation), while during the chronic phase number of deaths is lower (21 days after poisoning). Chronic phase of acute caustic poisonings is related to systemic complications and late post - corrosive complications (esophageal and gastric stenosis). Mortality and survival rates are higher in patients with more severe grades of post - corrosive injuries in the upper gastrointestinal tract, seen with upper endoscopy (grades ii b and iii). Admission of patients to the intensive care unit (icu) and their hospitalization in specialized hospitals are of a great importance for the final outcome . Our study showed that in patients with more severe post - corrosive damages in the upper gastrointestinal tract the mortality rate is higher and survival period is shorter . Duration of hospital stay and stay in the intensive care unit are positively correlated to the age of patients.
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In order to optimise survival in a temporally variable environment, many behavioural and physiological processes have evolved to have an optimally timed expression . These rhythmic processes may oscillate over the course of a year, a month, or a day and synchronise to the external environment in order to save energy at times when these processes would be inappropriate . Circadian clocks (from latin circa diem, about a day) influence almost all biological processes from sleep - wake rhythms, food intake, energy metabolism, body temperature, immune function, and cardiovascular function to cell proliferation and allow for accurate coordination of these processes with a period of approximately 24 h in the absence of external timing signals . Synchronisation of the circadian clock to the external environment occurs via stimuli such as light and food intake, the so - called zeitgebers (from german, time giver). It is currently accepted that the circadian regulation of physiology and behaviour occurs in a hierarchical manner, with a master clock residing in the paired structure of the suprachiasmatic nuclei (scn) in the anterior hypothalamus, which receives light input via the retinohypothalamic tract, and downstream subordinate clocks that occur in various tissues . In mammals at least, it is considered that most tissues and cells contain functional molecular clockwork similar to that of the scn, and within the hierarchical organisation of circadian timekeeping the adrenal gland plays a key role, since adrenocortical glucocorticoid rhythms are thought to synchronise clocks in various peripheral and central tissues [35]. According to the current model, the molecular circadian clock consists of interlocked transcriptional - translational feedback loops (ttls), with the positive arm being composed of the transcription factors circadian locomotor output cycles kaput (clock) or neuronal pas domain - containing protein 2 (npas2) and brain and muscle aryl hydrocarbon receptor nuclear translocator - like 1 (bmal1; also called arntl or mop3). These form heterodimers via pas domains to activate transcription of genes containing circadian transcription factor binding e - box elements including period (per 13) and cryptochrome (cry1/2), expressed in subjective daytime and comprising the negative arm of the core ttl . Per / cry complexes translocate to the nucleus where they accumulate over time and inhibit clock - bmal1 or npas2-bmal1 activity . In this way per and cry transcription is suppressed during subjective night - time, and the cycle completes as per / cry complexes are degraded towards the morning when clock / npas - bmal1 inhibition is released . Further, stabilizing ttls include the nuclear hormone receptors rev erb and rev erb and ror which regulate bmal1 expression and the basic helix - loop - helix transcription factors dec1 and dec2 [6, 7]. The hypothalamic - pituitary - adrenal (hpa) axis and glucocorticoids in particular have wide ranging effects on physiology and behaviour . Glucocorticoids are involved in the stress response, being secreted rapidly along with epinephrine under acute stress, and exert influence on metabolic functions such as glucose homeostasis, as well as immune and cognitive processes [811]. An interaction between the hpa axis and adipose physiology has long been proposed, and the effect of adipokines, adipose derived cytokines, on physiology, particularly with regard to metabolic disorders, is an area of active research . In modern industrial societies, life - style and work demands increasingly interfere with endogenously determined circadian rhythms . Off - shift moreover, work- or leisure - associated early or late wake times have led to the wide - spread phenomenon of social jet lag, where internal rhythms are overrun by artificial zeitgebers resulting in accumulation of sleep debt, for example, during the working week . Together this has led to an increased interest in the interaction of circadian rhythms and health parameters . Several metabolic disorders are associated with circadian disruption [1214], and obesity in particular is often accompanied by altered hpa axis rhythmicity [1518]. This review will discuss the circadian aspects of hpa axis regulation and adipokine secretion, their known interactions, and the potential consequences for human physiology . Glucocorticoids are key components of the hypothalamic - pituitary - adrenal (hpa) axis and act as the final effectors of this axis on other systems . Briefly, the production of the neuropeptides corticotrophin - releasing hormone (crh) and arginine vasopressin (avp) occurs at the paraventricular nucleus of the hypothalamus (pvn) under the circadian influence of the scn and stress - induced signals from the brainstem and the limbic forebrain [19, 20]. Both reach the pituitary via the hypophyseal blood portal system and stimulate corticotrophin (adrenocorticotropic hormone (acth)) secretion into the circulation . Acth in turn stimulates the production of glucocorticoids at the adrenal cortex, and glucocorticoids provide negative feedback by inhibiting the production of crh at the pvn and acth at the pituitary . Steroidogenesis occurs at the adrenal cortex where acth binds to the melanocortin 2 receptor (mc2r) leading via camp - pka signalling pathways to the transcription of steroidogenic genes such as cholesterol side chain cleavage enzyme (cyp11a1) and steroidogenic acute regulatory protein (star). In turn the key rate limiting stage of glucocorticoid production occurs with the transportation of cholesterol into adrenocortical cells via scavenger receptor class b member 1 (sr - b1) and low - density lipoprotein (ldl) receptors, and into the mitochondria by star . The rhythmic release of glucocorticoids into the circulation occurs in a robust circadian fashion under nonstressed conditions . Such is the robustness of the circadian cortisol rhythm in humans, that it has been used as a marker for the general circadian health of an individual [2224]. Circadian glucocorticoid rhythms peak slightly before the onset of the active phase in the late light phase for nocturnal species (e.g., rats and mice) and in the late dark phase for diurnal species including humans . A true circadian rhythm, this glucocorticoid secretion pattern persists in a constant environment and relies on an intact scn, first demonstrated in rats [26, 27]. The circadian pattern is overlaid by an ultradian rhythm consisting of pulses averaging between 80 and 110 minutes in humans and 50 and 60 minutes in rats . This ultradian rhythm has been demonstrated in vivo to be independent of the scn or any connection between the hypothalamus and pituitary [28, 29] and is controlled by negative feedback whereby glucocorticoids signal at pituitary glucocorticoid receptors (grs) to suppress acth secretion . The gr (also called nr3c1) is expressed throughout the body but is absent in the scn and mediates the acute effects of glucocorticoids which bind only during ultradian pulse peaks and rapidly dissociate from this receptor . In addition, glucocorticoids also bind to the mineralocorticoid receptor (mr, sometimes called the corticosterone receptor) with high affinity, which has a more specific tissue distribution, allowing for a permissive or long - term activation during the peak of circadian glucocorticoid concentration . There is evidence for the involvement of the mr in adipose tissue function, and glucocorticoids in particular are thought to act via this receptor to modulate adipogenesis . In addition to glucocorticoids, the mineralocorticoid aldosterone, important in the regulation of blood pressure, is known to have a circadian rhythm and act via the mr [34, 35]. Mice lacking bmal1 are hypotensive and in mice lacking both cry1 and cry2 the aldosterone rhythm is lost, being constantly high, and under a high salt diet these mice become hypertensive . The circadian regulation of glucocorticoid release is not only controlled by the hpa axis but is under the influence of several factors . Aside from influencing the hypothalamic component of the hpa axis, the scn also exerts its effects on the adrenal via the autonomic nervous system (ans), and furthermore, the adrenal gland is in possession of its own circadian clock . Within the hpa axis, circadian rhythms exist in the concentration of circulating acth and crh expression in the pvn, but these rhythms do not synchronise well enough to explain the regulation of rhythmic glucocorticoid concentration [38, 39]. The importance of acth for circadian glucocorticoid secretion should not be completely discounted, however, since acth can cause a phase dependent phase delay in adrenal glucocorticoid rhythms in vitro, indicated in tissue from mper::luciferase knockin mice, and acth can directly stimulate bmal1 and per1 expression in human explant adrenals . In their 2014 study, yoder and colleagues were able to stimulate a phase delay only, and so any effect of acth is independent of entrainment to the light cycle . Furthermore, the phase delay only occurred when stimulated at circadian time (ct) 18 in their experiments, and so although unlikely to be the main entrainment factor, acth may play a role in resetting the adrenal rhythm under certain conditions; that is, a stress response occurring at this vulnerable time may stimulate a phase delay of adrenal clocks . The scn exerts autonomic control over glucocorticoid rhythms via preautonomic pvn neurons projecting to sympathetic preganglionic intermediolateral neurons of the spinal cord and splanchnic nerve innervation of the adrenal . The importance of the autonomic influence on glucocorticoid rhythms has been demonstrated in hypophysectomised rats [45, 46] and by splanchnic nerve transections [47, 48]. The mechanism of ans stimulation of glucocorticoid rhythms remains to be elucidated and is reviewed in greater detail elsewhere . Preceding the description of the molecular circadian clock, a robust circadian rhythm of steroid secretion was first demonstrated in isolated adrenal glands of the syrian hamster, and more recently, rhythmic expression of clock genes has been well demonstrated in the adrenal cortex of both rodents and primates [5156]. Approximately, 10% of the murine adrenal transcriptome shows circadian variation, including genes involved in cholesterol transport, steroidogenesis, and acth signalling . Mice lacking genes from the negative arm of the ttl are hypercortisolic [5, 57] and in contrast, those lacking genes encoding bmal1 or clock are hypocortisolic [58, 59]. Clock deficient mice lack the rhythmic expression of star, being constantly high in per1/2 and cry1/2 mice, while star shows reduced expression in bmal1 mice . The relative importance of the central and local adrenal circadian clock in the regulation of glucocorticoid rhythm is an area of active research . The adrenal clock appears to have an important role to play in the regulation of adrenal acth sensitivity, being rhythmically regulated by a gating mechanism via the local circadian clock . In consequence, adrenals from clock - deficient mice without functioning per2 or cry1 have defective corticosterone synthesis when transplanted to wild - type adrenalectomized hosts . The importance of star as a link between the molecular clock and steroidogenesis has been demonstrated by the overexpression of clock and bmal1 in the mouse adrenocortical y1 cell line, which led to increased star expression and steroid production, and which was then inhibited upon application of antisense star oligodeoxynucleotides . In the same study, son and colleagues were able to demonstrate that an adrenal conditional knockdown of bmal1 led to loss of star and intra - adrenal corticosterone rhythmicity in the absence of a light cue . Ex vivo cultured adrenals from mice lacking bmal1 have blunted corticosterone secretion in response to acth . Similarly, in explant studies on primate adrenal tissue, the knockdown of cry2 and subsequent loss of bmal1 expression were accompanied by blunting of acth stimulated increases in cortisol secretion as well as star and 3 - hydroxysteroid dehydrogenase protein expression . The hpa axis, through glucocorticoids, exerts influence on many important biological processes, and glucocorticoids are proposed to have an important synchronizing role on peripheral circadian rhythms [3, 63]. Glucocorticoids have been found to broadly influence gene expression through gr . When activated, the cytoplasmic gr, previously in an inactive complexed state, undergoes conformational changes and, after dimerization, translocates into the nucleus . In the nucleus gr activates transcription of glucocorticoid target genes by binding to glucocorticoid response elements (gres). These gres regulate expression of several genes including some core clock genes such as per1, per2, npas2, and rev - erb [3, 4, 66, 67]. The capability of glucocorticoids to shift the rhythm of peripheral clocks has been demonstrated in vitro and in vivo with dexamethasone, a synthetic glucocorticoid analogue, inducing clock gene and clock dependent gene expression in rat fibroblasts . Furthermore, on administration to live mice dexamethasone was able to delay or advance the phase of clock gene expression in the liver, kidney, and heart depending on the time of injection . More recently, evidence for the effect of glucocorticoids to influence circadian rhythms of human adipose tissue has also been provided . In addition, in vitro studies indicate that molecular clock components are able to negatively regulate the action of gr to influence gene expression . Clock / bmal1 is able to physically interact with gr to inhibit binding to gre sites in order to suppress glucocorticoid stimulated gene expression in human colon cancer hct116, and human cervical cancer hela cells . Similarly in vitro and in vivo murine studies demonstrate the importance of cry1 and cry2 for suppression of glucocorticoid stimulated gene expression since both cry1 and cry2 are able to interact with the gr in order to oppose activation of this receptor . Desynchrony between the central scn clock and peripheral oscillators can be brought about by inappropriately timed food intake . Under normal conditions in mice, feeding is largely restricted to the dark phase . By restricting food access to the light phase, core clock genes such as per1, per2, per3, reverb, and cry1 and clock output genes such as dbp and cyp2a5 are phase shifted by 812 h in the liver independent of scn per1/2 expression . In the same study, phase shifts were additionally observed for dpb expression in the kidney, heart, and pancreas, while mice fed exclusively in the night had peripheral rhythms similar to those fed ad libitum . These shifted peripheral clock rhythms correlate with changes in glucocorticoid signalling, with daytime feeding in mice causing an additional peak in circulating corticosterone in advance of the peak observed at 12 h following lights on (zeitgeber time; zt 12) for ad libitum fed animals, and the phase shift in the liver was absent in mice lacking gr in this organ . Outside the experimental context, timing of food intake can be influenced by social or environmental factors such as shift work, sleep curtailment, or inter - time - zone travel (jetlag). Interestingly, disruption of diurnal feeding rhythms can also be induced by high - fat diet, with mice roughly doubling the proportion of nutrient intake during the light phase after only one week . This alteration of diet is accompanied by dampening of clock gene expression rhythms in liver and fat tissue and altered rhythms of several circulating metabolic factors including corticosterone and leptin . Thus, the timing of food intake, whether caused by environmental factors or influenced by diet composition, is likely important for the maintenance of peripheral and central clock synchrony . Obesity is associated with a dampened circadian glucocorticoid rhythm in wild type and in genetically obese rodents [7375] and humans [1518]. A correlation between the abdominal fat distribution, elevated dietary lipid (and in particular higher saturated fatty acid) content, and disturbance of the hpa axis has been found in women, who have a low variability between morning and evening salivary cortisol . In the same study, women with less difference between morning and evening cortisol samples, as well as preferring food containing on the other hand, mice with a genetically disrupted circadian clock display disrupted feeding rhythms and high propensity to metabolic disease [59, 77, 78]. Per1 mutant mice have constantly high corticosterone levels and fail to gain weight as efficiently as wild type animals despite the high body weight - adjusted food intake, suggestive of increased metabolic rate, along with increased glucose metabolism that the authors attribute to the lack of a robust glucocorticoid rhythm . In clock mutant mice, several genes important for appetite regulation lose rhythmic expression in the hypothalamus . This is associated with a strongly attenuated diurnal feeding rhythm as well as increased weight gain on both standard and high fat diet, going along with measurable detrimental effects on circulating metabolic parameters including glucose, cholesterol, triglyceride, and leptin levels . In the cry1/cry2 double - deficient mouse, as previously discussed, these mice are hypercortisolic and have impaired glucose metabolism . When kept on a high fat diet, besides gaining more weight, these mice also show increased weight gain relative to food intake, associated with a loss of rhythmicity in metabolic rate, increased fat mass, and insulin secretion . This is in contrast to mice lacking only cry1, however, that are more resistant to diet - induced obesity and show decreased overall fat mass compared to wild type controls . In the same study, griebel and colleagues found no difference between cry2 knockout mice and wild type controls in terms of the response to high fat diet interestingly, glucocorticoids may play a role in adipocyte differentiation via the mr, with knockdown of this receptor, but not gr, inhibiting adipogenesis in the murine 3t3-l1 cell line . Quantitatively, most white adipose tissue (wat) is of either subcutaneous or visceral origin in humans . While subcutaneous depots mainly store energy and provide thermal insulation, internal wat depots have a higher endocrine activity . White adipocytes are long - term energy stores and accumulate energy in the form of triglycerides (tgs), which are either absorbed directly from the bloodstream or generated within the adipocyte by de novo lipogenesis . In the converse process of lipolysis, white adipocytes break down tgs and release the resulting free fatty acids (ffas) and glycerol into the circulation in order to support other organs during times of energy shortage or acute stress situations . In order to prevent metabolic disorders, lipogenesis and lipolysis in white adipocytes are both tightly regulated since high concentrations of circulating ffas and tgs can cause lipotoxicity and promote cardiovascular complications, and hyper - uptake of tgs can result in undue increase of adipose tissue mass . Therefore, these processes have evolved to be regulated not solely in response to food intake, but are under control of clock - mediated circadian rhythms [83, 84]. Local adipose transcription rhythms and a number of genes encoding key lipogenesis and lipolysis enzymes have promoters that harbour e - box elements, and thus are direct targets of the circadian clock . Of more than 2,000 genomic loci in mouse liver that have been identified to be directly rhythmically regulated by bmal1 and clock using chromatin immunoprecipitation with parallel dna sequencing (chip - seq), many encode genes which are involved in carbohydrate metabolism (e.g., glut2, pck1, and gys2) and lipid metabolism (e.g., dgat2, lipe, and pnpla2). It is still to be determined whether a similar extent of these bmal1/clock targets can be found in adipose tissue; however, it is known that bmal1 directly and rhythmically controls the transcription of lipe and pnpla2 in wat . In addition, robust and coordinated expression of circadian oscillator genes (npas2, bmal1, per13, and cry1 - 2) and clock - controlled downstream genes (rev - erb, rev - erb, dbp, e4bp4, stra13, and id2) has been described for several murine adipose tissues . Furthermore, the molecular clock can be linked to lipid metabolism since per2 directly and specifically represses the activity of peroxisome proliferator - activated receptor gamma (ppar), a nuclear receptor critical for adipogenesis, the regulation of insulin sensitivity, and inflammatory responses . Thus, per2 is proposed to have an important role to play in the regulation of ppar-mediated adipogenesis . The circadian timing system regulates the rate of lipid storage and mobilisation over the course of the day to ensure optimal energy supply and metabolism . The action of adipose tissue is dictated by the active and rest phases of the daily cycle . In humans this means that storage and lipogenesis both occur during the daytime when active food intake allocates energy demands, and reciprocally, the release of glycerol and ffas is predominantly restricted to the night . In nocturnal mammals, as with most rodents, this process is accordingly reversed . Adipokines are peptide hormones that are produced in and secreted from adult adipocytes, regulating diverse biological processes and underlying the key role of adipose tissue in the regulation of energy homeostasis [8890]. As with lipid mobilisation, the secretion of numerous adipokines is under circadian control (reviewed in), with leptin and adiponectin being the most prominent adipocyte derived peptide hormones with a distinct diurnal rhythm . These adipokines have diverse actions; they are involved in metabolic regulation and control important aspects of lipid metabolism, glucose disposal, and adipose endocrine function [9294]. The rhythmicity of adipokine secretion appears to rely on intrinsic circadian oscillators, a combined control via the master pacemaker in the scn and local control at the level of adipocyte clocks . Leptin can act directly at the hypothalamus, the main region of energy homeostasis regulation in the brain, to increase energy turnover and inhibit appetite . Irrespective of diurnality or nocturnality, for mice, rats, and humans, leptin concentration peaks at night . In humans this is during the normal rest phase, when hunger is suppressed and metabolic turnover in adipose tissue sustains energy supplies . Disrupted leptin secretion is associated with night eating syndrome, an eating disorder where the patient's sleep cycle is disrupted and significant quantities of food are consumed during the night [96, 97]. Altered leptin concentrations can be also observed in shift workers and this is associated with diminished satiety and obesity . A large part of the metabolic activity of adipose tissue relies on the interaction of leptin, adiponectin, and insulin . Adiponectin is considered important for the modulation of cellular glucose uptake, adipocyte insulin sensitivity, and fatty acid break down [99, 100]. While in lean patients the blood content of adiponectin and leptin oscillates inversely, the adiponectin concentration in obese patients is decreased while leptin is elevated . The expression of gr in white adipose tissue also oscillates with a diurnal rhythm . Under unstressed conditions the mrna expression of gr in rat wat peaks at the transition from the light / inactive to the dark / active period just as in humans the highest concentration of circulating cortisol cortisone reductase (also known as 11-hydroxysteroid dehydrogenase type 1; 11hsd-1) is the key enzyme that locally regenerates the inactive form of 5-tetrahydrocortisol (humans) or 11-dehydrocorticosterone (rodents) [103105]. While its expression in rat hippocampus oscillates in a circadian manner, rhythms of 11hsd-1 in peripheral tissues could not be determined . The expression of 11hsd-1 is locally controlled and is important for amplifying glucocorticoid feedback to the hpa axis, in addition to influencing glucocorticoid action . Under obese conditions hippocampal oscillation is dampened in rats, and with a disturbed hpa axis function the local 11hsd-1 activity and gc action are also altered [104, 105]. As with the majority of peripheral rhythms investigated, the intrinsic adipocyte clock is synchronised by the scn via neuronal and endocrine pathways . These scn pathways to the periphery overlap closely with that of the hpa axis and enable potential interactions . Disrupted hpa axis rhythms are associated with the obese state and, in humans, obesity is correlated with increased glucocorticoid production and degradation, resulting in an overall higher cortisol turnover rate . Furthermore, under stressed or hypocortisolaemic conditions, modulation of adipocyte and adipokine circadian rhythm is likely to occur . Indeed, gre regions have been found in the promoter regions of several genes involved in adipocyte function, including triglyceride accumulation and adipogenesis . Moreover, human explant visceral and subcutaneous adipose tissue clock gene expression rhythms can be altered by dexamethasone administration . In light of this, an interaction pathway with the hpa axis to mediate food intake and body weight via the circadian output of adipocytes is postulated . Since chronic stress is correlated with disrupted or dampened rhythmicity in adipose function, this may increase the propensity of weight gain and adiposity further, promoting the development of metabolic disorders such as diabetes under long - term stress exposure . In contrast, the stress response is altered in different metabolic states and in particular is hyperresponsive under obese conditions . Leptin is the best studied of all adipokines to date, and although this is an area of active research, the majority of evidence for adipokine interaction with the hpa axis exists for this peptide . In the healthy state, glucocorticoids are generally considered catabolic and are known to strongly affect leptin expression . In vitro glucocorticoid application to isolated adipocytes stimulates the synthesis and secretion of leptin [112115]. In rats, the peripheral infusion of glucocorticoids induces ob (leptin) gene expression in adipose tissue and overall hyperleptinaemia, resulting in decreased food intake and a reduction in body weight [116, 117]. This is also true for humans, with peripheral administration of glucocorticoids increasing leptin secretion . Therefore, it can be suggested that an impaired adrenal function and resulting hypocortisolemia leads to lower leptin expression in humans and rodents [118124]. Direct hypothalamic leptin application generates a similar effect on food consumption as reviewed by friedman and halaas, suggesting that peripheral glucocorticoids may drive leptin mediated appetite effects . In contrast, constant (and therefore arrhythmic) central infusion of glucocorticoids has been demonstrated to increase food intake and body weight, with concurrent hyperleptinaemia, hyperinsulinaemia, and hypertriglyceridaemia that occur in obesity, possibly mediated by altered hypothalamic expression of neuropeptide y and crh as proposed for rats . Therefore, a central stimulatory effect of glucocorticoids on food intake can be hypothesised and counter - regulated by elevated leptin levels, while central actions of leptin might be inhibited under glucocorticoid influence . It should be noted that the effect of glucocorticoid action on other factors such as insulin secretion might also play a role in mediating hpa axis effects on energy balance . However in patients with cushing's disease, characterised by pituitary or adrenal adenoma, hypercortisolemia, and disrupted cortisol rhythms, leptin levels are highly independent of adiposity, and removal of the tumour usually results in lowered levels of both cortisol and leptin [126, 127]. In contrast, adrenalectomized rats experience potent effects on food intake and body weight when leptin is administered, and this effect is inhibited by dexamethasone administration . Taken together this suggests that high levels or disrupted blood rhythms of glucocorticoids may contribute to the leptin resistance observed in obesity . Of note, patients with cushing's disease develop increased visceral adiposity, while abdominal subcutaneous fat depots are depleted . This suggests that the impact of glucocorticoids on different kinds of adipose tissue varies enormously . Interestingly, leptin deficient ob / ob mice show an intact circadian glucocorticoid rhythm despite an overall raised circulating concentration in contrast to the db / db mouse, which lacks a functional leptin receptor and along with being hypercortisolaemic it also displays disrupted glucocorticoid rhythms . A fasting induced increase in circulating glucocorticoid and acth concentration similarly, the high levels of glucocorticoids observed in ob / ob mice can be rescued by leptin substitution [73, 132]. A direct adipo - adrenal feedback loop has been postulated [133135], with leptin being the suppressive arm and the hpa axis, specifically glucocorticoids, being the positive arm . The mechanism of action for leptin suppression of glucocorticoid concentration may be located at the level of the hypothalamus, since crh secretion from isolated hypothalamic neurons is inhibited by leptin, but this is not true for acth secretion from isolated pituicytes . Leptin is also capable of interacting directly with the adrenal gland, with functional leptin receptors being present in the adrenal cortex and to a lesser extent in the catecholamine - producing adrenal medulla [136, 137]. Furthermore, the leptin response is absent in adrenal cells from db / db mice . Leptin has been found to inhibit basal and acth stimulated secretion of glucocorticoids in rodent and human adrenal tissue [136, 138, 139], with additional effects on the secretion of aldosterone in human adrenals being reported (figure 1). Taken together, these data suggest a regulatory role of leptin on the hpa axis, and although this interaction is unlikely to directly drive the circadian rhythm of glucocorticoids in circulation, leptin may modulate this rhythm at the level of the hypothalamus or the pituitary . This is of particular interest in the context of metabolic disease, since altered leptin profiles (such as leptin resistance, insufficient or dysfunctional leptin production or signalling) accompanying obesity may interact with the hpa axis in order to contribute to metabolic disorder . Interactions between the hpa axis and adiponectin have also been described, although the effects appear contradictory and therefore a clear relationship is controversial . Glucocorticoids and acth both suppress adiponectin secretion from wat in human and murine cell culture experiments [140, 141]. Reciprocally, adiponectin receptors have been observed throughout the rodent and human adrenal gland [142, 143]. Unlike leptin, adiponectin (adipoq) mrna has further been detected in the adrenal gland, in the zona glomerulosa of the adrenal cortex in rats, although this is in contrast to studies in mice and the murine adrenocortical y-1 cell line . In vivo administration of excess glucocorticoids to rats reduces circulating adiponectin concentrations, and adrenalectomy leads to a reduction of adiponectin gene expression in epididymal wat . The reported effects of adiponectin on glucocorticoid secretion are also contradictory . For example, in vitro administration of adiponectin to adrenocortical y-1 cells suppresses basal and acth stimulated glucocorticoid secretion and concordant alteration of steroidogenic gene expression, including that of star and cyp11a1 . However, in a different study, adiponectin administration to a primary culture of rat adenocytes led to dose - dependent enhancement of corticosterone secretion . Several inflammatory cytokines are produced and secreted as part of the adipokine function of adipose tissue . Interleukin-6 (il-6), tumour necrosis factor (tnf-), and chemerin secretion from adipose tissue fluctuate diurnally, with rhythms peaking during the rest phase, which is daytime for rodents [146148] and during the late night or early morning for humans . Glucocorticoids, in line with their immunosuppressive function, counteract on this release to minimise damage of host tissue [150, 151]. Research of the last decades has uncovered a tight interaction of the circadian rhythms of the hpa axis, adipocyte function, and adipokine secretion (figure 1). The integration of these systems is essential in the maintenance of health, and the deregulation or alteration of one rhythm, in the context of disease or by lifestyle interventions, is likely to be reciprocated in the other systems . For example, obesity is correlated with hypercortisolemia and altered hpa axis rhythmicity and we have described the reciprocal effects of alterations in these rhythms above, thus increasing the vulnerability for hpa axis disorders and vice versa . Thus, stabilising circadian glucocorticoid rhythms may be an important approach to counteract metabolic disorders . Under acute stress, glucocorticoid release feeds back to the hypothalamus to initiate the return to homeostatic conditions, and at the same time, mobilizes energy from body stores . Irregular sleep / wake cycles, jetlag, or shift work can lead to a constant disruption of circadian processes very much resembling the effects of chronic stress exposure . A persistently activated hpa axis results in elevated blood glucose, hyperinsulinaemia, and insulin and leptin resistance . Obesity is accompanied by an adipose induced systemic inflammatory state which may contribute to the development of rheumatoid arthritis . Obesity - associated dampened physiological and behavioural rhythms can further affect sleep quality, food intake, and immune function [156158]. In rheumatoid arthritis patients, long - term administration of high doses of hydrocortisone can lead to cushing's disease - like phenotypes including diabetes mellitus, osteoporosis, hypertension, dyslipidemia, and sleep disorders . To restore disrupted endocrine glucocorticoid rhythms, timed substitution or inhibition of steroid levels is required . In hypocortisolaemic disorders exogenous hydrocortisone administration is the mainstay of treatment, primarily serving to restore adequate stress responses and bypass the symptoms of chronic adrenal insufficiency such as weight loss, fatigue, and nausea . On the other hand, hydrocortisone doses should be minimised to avoid adverse side effects of glucocorticoid surplus such as weight gain, osteoporosis, or impaired glucose tolerance . In an attempt to mimic physiological glucocorticoid patterns the total dose (between 15 and 25 mg per day) is distributed into two or three quantities with the highest dose applied during the morning . Timing the last hydrocortisone dose at least 5 - 6 h before bedtime prevents sleep disruption due to supra - physiological cortisol concentrations . An alternative approach is a dual - release hydrocortisone preparation, which is currently tested in patients with adrenal insufficiency [165167]. A profiled hydrocortisone infusion controlled by a subcutaneous pump has been tested in a small cohort of addison patients, and another approach used a delayed release formulation of prednisone which, when taken in the evening, leads to an increasing availability of the drug starting at approximately 2 a.m. to counteract morning stiffness in rheumatoid arthritis patients . The effect of such chronotherapeutic approaches on adipose function, however, remains to be shown . Another yet unexplored route, that would rather target hypercortisolaemic conditions, as seen in cushing's disease or stress disorder patients, involves timed suppression of glucocorticoid levels, for example, by the antisteroidogenic drug metyrapone . In mice, this has been shown to alleviate jetlag - induced disruption of the rest - activity cycle . While the circadian rhythm of glucocorticoid secretion was among the first described, adipose rhythmicity and adipose clocks have raised more recent interest because of their pivotal role in energy homeostasis . By release of endocrine factors (glucocorticoids and adipokines) chronotherapeutic approaches targeting this crosstalk may be fruitful in treating metabolic diseases and stress disorders . It is tempting to speculate that this may not be restricted to targets in peripheral tissues but may extend to central aspects of appetite regulation and stress- and obesity - associated neuropsychiatric alterations.
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These include human population density and behaviour, housing type and location, water supply, sewage and waste management systems, land use and irrigation systems, availability and use of vector control programmes, access to health care, and general environmental hygiene . Meteorological factors that influence transmission intensity of infectious diseases include temperature, humidity, and rainfall patterns . Social and demographic factors such as population growth, urbanization, immigration, changes in land use and agricultural practices, deforestation, international travel, and breakdown in public health services have been mainly responsible for the recent resurgence of infectious diseases1 . The intergovernmental panel on climate change noted in its 2007 report that climate change may contribute to expanding risk areas for infectious diseases such as dengue and may increase the burden of diarrhoeal diseases, putting more people at risk2 . Global climate change is a phenomenon that is now considered strongly associated with human activities . Atmospheric carbon dioxide levels, which have remained steady at 180 - 220 ppm for the past 420,000 years, are now close to 370 ppm and rising3 . Due to improvements in meteorology, we are now able to better understand long - term changes in climate . Such understanding might enable the prediction of where and when infectious disease outbreaks may occur . Box 1 outlines some of the consequences of climate change that clinicians in india could expect to see over the coming years . We describe in this paper these links between climate and health, future implications, and recommendations for clinician - patient interaction . What clinicians might expect due to climate change the effects of climate change on human health in india is a broad topic, covering areas from extreme weather events to shifts in vector - borne diseases . In south asia, scientists predict an increased frequency of floods due to greater intensity of rainfall events and to glacier lake outburst floods (glofs) in mountainous regions5 . These trends are already being seen . In 2007, floods resulting from monsoon rains killed more than 2,000 persons and displaced more than 20 million persons in bangladesh, india, and in nepal6 . In the himalaya region of south asia, the frequency of glofs rose during the second half of the 20 century, and glofs have occurred recently in nepal, india, pakistan, and bhutan78 . For example, if floodwaters become contaminated with human or animal waste, the rate of faecal - oral disease transmission might increase, allowing diarrhoeal disease and other bacterial and viral illnesses to flourish . Faecal - oral transmission of diseases is of particular concern in regions such as south asia because of limited access to clean water and sanitation . In developed countries, flood control efforts, sanitation infrastructure, and surveillance activities to detect and control outbreaks minimize disease risks caused from flooding9 . In developing countries, increase in diarrhoeal disease, cholera, dysentery, and typhoid is of specific concern10 . For example, after flooding in west bengal in 1988, cholera was thought to be the cause of an outbreak of diarrhoea that resulted in 276 deaths11 . Numerous studies have linked previous floods in bangladesh and parts of india with outbreaks of diarrhoea as well as respiratory infections12131415 . Flooding can also contribute to increased vector - and rodent - borne and other infectious diseases . For example, collections of stagnant water provide breeding grounds for mosquitoes, potentially aiding in the spread of malaria16 . Other studies have linked flooding in bangladesh and parts of india with outbreaks of rotavirus and leptospirosis1718192021 . Rising sea - surface temperatures although cyclones originating in the bay of bengal and the arabian sea have decreased in frequency since 1970, these have increased in intensity, causing significant damage in india and bangladesh222324 . Public health effects of cyclones include diseases and illnesses associated with the loss of clean water, hygiene, and sanitation, loss of shelter and belongings, population displacement, toxic exposures, and hunger and malnutrition risk due to food scarcity25 . A warmer climate could cause water - borne diseases to become more frequent, including cholera and diarrhoeal diseases such as giardiasis, salmonellosis, and cryptosporidiosis9 . Diarrhoeal diseases are already a major cause of morbidity and mortality in south asia, particularly among children . It is estimated that one - quarter of childhood deaths in south asia are due to diarrhoeal diseases26 . As rising ambient temperatures increase, bacterial survival time and proliferation and thus the incidence of diarrhoeal diseases might further increase27 . Diarrhoeal diseases are largely attributable to unsafe drinking water and lack of basic sanitation; thus, reductions in the availability of freshwater are likely to increase the incidence of such diseases28 . Rapid urbanization and industrialization, population growth, and inefficient water use are already causing water shortages in india, pakistan, nepal, and bangladesh6 . Climate change will exacerbate the lack of available fresh water as annual mean rainfall decreases in many areas . Cholera is a well - known water - borne diarrhoeal disease that has afflicted humankind since ancient times . Outbreaks of cholera have occurred in india, bangladesh, and more recently, latin america and africa29 . Molecular techniques have shown that bacteria are now recognized as naturally occurring in aquatic environments, with bacterial population peaks in spring and fall in association with plankton blooms29 . The discovery of vibrio cholerae in the natural environment, with a dormant state between epidemics, changed the understanding that this disease had only a human reservoir . A relationship has been observed between increase in sea - surface temperature and the onset of cholera epidemics, with the cholera outbreaks following the seasonal rise and fall in sea - surface height and temperature29 . Increases in cholera bacterial populations associated with plankton blooms in spring and summer have been noted off the coasts of several latin american countries and in bangladesh29 . About 400 - 500 million cases of malaria and more than 1 million malaria - related deaths occur globally each year3 . Several factors have caused the global resurgence of malaria, including the emergence of insecticide and drug resistance, human population growth and movement, land - use change, and deteriorating public health infrastructure . Changes in temperature, rainfall, humidity, and immunity levels also affect malaria transmission . All these factors can interact to affect adult mosquito densities and the development of the plasmodium parasite within the mosquito . Both the parasite and the mosquito that houses the parasite are susceptible to temperature changes . At temperatures close to the physiological tolerance limit of the parasite, a small increase in temperature can kill the parasite, thereby decreasing malaria transmission . At lower temperatures, however, a small increase in temperature can greatly increase the risk of malaria transmission due to increased numbers of mosquitoes3 . For example, deforestation might elevate local temperatures and changes in housing types might change indoor temperatures where vectors spend most of the time resting . Deforestation, vegetation clearance, and irrigation can form open pools preferred by malaria vectors and thus increase transmission . In south asia, malaria is one of the most pertinent examples of increased occurrence of a vector - borne disease . Malaria is already one of the most important vector - borne diseases in india, bangladesh, and sri lanka; changes in temperature and precipitation patterns have the potential to expand the geographical range of malaria into temperate and arid parts of south asia9 . For example, in india malaria distribution is expected to expand to higher latitudes and altitudes . Because the relationship between climate and disease distribution is complex, in some areas reductions in transmission intensity in endemic areas might lead to greater proportions of the population losing immunity, resulting in epidemics in later years30 . Currently, all of india's population is at risk for contracting malaria except for those in the areas above 1700 m above sea surface . More than 973 million persons are exposed to vector - borne malarial parasites in india, and in 1998 an estimated 577,000 disability adjusted life years (dalys) were lost due to malaria31 . The multifactorial nature of malaria causation does not lend itself easily to a simple model of disease prediction . To determine the role of climate change in malaria transmission, research efforts will be required that incorporate a disease surveillance system combining trend analyses from multiple sites to account for local factors . The system combined human and meteorologic factors to provide about a month's lead - time of epidemic risk . Rainfall alone accounted for about 45 per cent of the variation in malaria transmission32 . By the 2050s, the geographic range of malaria vectors the duration of the transmission window is likely to widen in northern and western states and shorten in southern states . Malaria is likely to persist in orissa, west bengal, and southern parts of assam . It might shift from central india to the southwestern coastal states (maharashtra, kerala, and karnataka). The northern states might also become prone . The duration of exposure is likely to widen in north and west india, and shorten in south india33 . These include mosquito - borne diseases such as chikungunya fever and dengue, parasitic diseases such as leishmaniasis, lymphatic filariasis and onchocerciasis, and tick - borne diseases, which may exhibit changes in transmission intensity or shifts in their geographical ranges due to the impact of climate on the relevant vector populations . Climatic factors might also influence human plague, a bacterial disease carried by rodents and transmitted by fleas . Temperature and rainfall are important determinants of rodent population abundance and distribution . Combined with the influence of temperature and humidity on flea survival and development, changes in any of these climatic components may result in changes in plague incidence30 . Murine typhus, a rickettsial disease, is also transmitted by fleas and thus may exhibit similar climate sensitivity30 . The arboviral diseases chikungunya and dengue may also be influenced by climate, as both are transmitted by the common vector aedes aegypti . The first reported outbreak of chikungunya in india was in 1963 in calcutta (now kolkata), with transmission continuing until 197334 . The virus reemerged in 2005, and has since spread rapidly, with more than one million cases reported - despite no standardized surveillance system for the disease3435 . Dengue has also been a significant problem, with more than 50 dengue outbreaks reported in india since 196036 . The activity, abundance, distribution, and ability to transmit viruses is influenced by temperature and precipitation30 . As a developing country with high population density, india might experience myriad human health effects because of climate change . These effects could include infectious diseases such as malaria, chikungunya, and water - borne illnesses . Monitoring the spread of infectious diseases will require early warning systems, which have both health and economic benefits . Displacements due to the loss of housing, hunger, and injuries are some of the adverse outcomes to the population . Health care providers will need to address at the primary level the negative health outcomes associated with climate change in india . There will likely be a rise in demand for emergency medicine services such as urgent and ambulatory care37 . Expanded surveillance activities could detect shifting patterns of disease distribution so that emergency department personnel would be aware of emerging threats . Many other fields of health care in india could be impacted by climate change, including family practice, internal medicine, paediatrics, geriatrics, and psychiatry37 . The climate and health council of the general medical council of the united kingdom38 has outlined several actions that health care providers can take, many of which are relevant to providers in india39 . These include informing professional colleagues and the wider community about the health consequences of climate change, and advocating for carbon reductions to promote human health . Several recommendations have been made for health system preparedness, some of which are relevant for indian hospitals39 . These include undertaking energy audits, pursuing energy and water conservation and energy efficient construction . A review of overall emissions from petroleum - based energy sources such as diesel generators and hospital transportation systems is a key first step . Contingency plans for alternative methods of energy generation during electricity blackouts, particularly during the summer months, will facilitate preparedness for these events . To reduce waste, health care without harm has recommended several options: recycle and buy recycled products, collect and recycle nitrous oxide and anaesthetic gases, prevent waste, and dispose of waste locally40 . Using native vegetation and planting trees on site can mitigate the heat - island effect . Vegetation lowers ground level temperatures near buildings, reducing the buildings cooling load, energy requirements, and greenhouse gas emissions . Those of low socio - economic status will likely to be the most affected by the health impacts of climate change, as they have the least adaptive capacity4 . As india's economy continues to expand, the growing middle class presents a unique situation . While rising out of poverty will improve sanitation levels and living conditions, thus increasing resilience to infectious diseases, it will also lead to higher consumption patterns that can initiate new health problems while leading to more carbon pollution . Box 2 outlines some of the topics health care providers can discuss with their patients to help maximize the benefits . General practitioners as well as specialists both can play a role in anticipating the health effects of climate change and improving the health through discussing effective preventive health care with their patients . Discussing climate change effects with patients
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The host immune system recognizes and eliminates invading pathogenic microorganisms such as viruses, bacteria, and fungi . Recently, the mechanisms by which the innate immune system recognizes pathogen have been extensively studied . Pattern recognition molecules / pathogen recognition receptors (prrs) are classified into three families: toll - like receptors (tlrs), rig - i - like receptors (rlrs), and nucleotide binding - oligomerization domain (nod)-like receptors (nlrs) [1, 2]. The rlr family contains retinoic acid - inducible gene i (rig - i) and melanoma differentiation associated gene 5 (mda5). The nlr receptor family contains nod1, nod2, nlrp3, nlrpc5, nlrp1, naip, and ciita . In addition, dna - dependent activator of interferon regulatory factors (dai) has been identified as a dna sensor . Various microbial components are recognized as their vague and common molecular shapes by prrs . Early responses against virus infection are initiated on recognition of pathogen - associated molecular patterns (pamps) by pattern recognition molecules, triggering two responses . One is the production of interferons (ifns) resulting in an antiviral state as part of the innate immune response, and the second is maturation of dendritic cells (dcs) to establish acquired immunity . In order to establish an infection within a host, viruses must escape from and/or suppress the immune system by various strategies . An important strategy used by viruses tlr signaling proceeds via two pathways: the myeloid differentiation factor 88 (myd88)-mediated pathway, and the toll - interleukin-1 receptor (tir)-domain - containing adaptor inducing ifn- (trif)-mediated pathway [1, 2]. The former causes activation of the transcription factor nf-b, which activates various genes contributing inflammatory reactions . The latter causes induction of ifns, whose stimulation leads cells to antiviral state . Tlr3 signaling activates irf-3, an important transcription factor for ifn-, and ifn production is induced . However, tlr4 activates both pathways, so tlr4 agonists activate nf-b and induce ifn production . Cytosolic prrs, such as rig - i, mda5, and dai, commonly activate irf-3 . The expression of prrs differs depending on the cell type . Importantly, it is different between cells derived from myeloid stem cells (myeloid dendritic cells (mdcs), monocytes, macrophages, langerhans cells, and neutrophils) and cells derived from lymphatic stem cells (plasmacytoid dendritic cells (pdcs), t cells, and b cells). Tlr7 and tlr9 are rarely expressed on mdcs, whereas tlr3 and tlr8 are rarely expressed on pdcs . Tlr4, on the other hand, is expressed at very low levels on both pdcs and mdcs . Initially, viruses invade the host epithelial tissues found in the oral cavity, respiratory tract, intestinal tract, and the urogenital apparatus (figure 1). Lamina propria dcs, langerhans cells, and stromal cells are resident in these tissues . In the connective tissues, fibroblasts resident, and capillary vessel and lymphatic vessel are expanded . Monocytes, macrophages, t cells, b cells, pdc, and mdc circulate within the blood vessels and lymphatic vessels, and patrol the interstitial spaces . Virus - infected epithelial cells and fibroblasts produce ifns, mainly ifn- and ifn-, which provide surrounding uninfected cells with antiviral state . Furthermore, chemokines and cytokines, such as interleukin-1 (il-1), il-6, il-8, granulocyte - macrophage colony - stimulating factor (gm - csf), and tumor necrosis factor- (tnf-) are also produced . These molecules promote chemotaxis of the resident dcs (lamina propria dcs and langerhans cells) toward virus - infected and dead cells . Neutrophils, monocytes, macrophages, plasma cells, mdcs, and pdcs also migrate from the blood vessels to the site of infection . Ifn--inducible protein 10 (ip-10), monocyte chemotactic protein 1 (mcp-1), macrophage inflammatory protein-2 (mip-2), mip-3, and mip-3 largely contribute to the transmigration of blood dcs . However, these blood - borne immune cells can also be infected by viruses, which can then modulate the production of various cytokines and chemokines . To establish an infection, viruses need to suppress a number of host immune responses, the antiviral activity induced by ifns, the chemotaxis of immune cells induced by chemokines / cytokines, the maturation and activation of dcs, activation of nk and nkt cells, transmigration of mature dcs to the lymph nodes, and the differentiation and activation of t cells and b cells in the lymph nodes, for example . When viruses infect immune cells, such as dcs, the infected cells frequently show suppression of maturation and differentiation, suppression of cytokine receptor and costimulatory molecule expression, secretion of molecules that mimic cytokines and cytokine receptors, and so on . These strategies are used by the virus to inhibit the acquired immune response . In addition htlv-1, which is a retrovirus, infects cd4 t lymphocytes, cd8 t lymphocytes, dcs, b cells, macrophages, and astrocytes, and preferentially replicates in cd4 t lymphocytes . Htlv-1 causes latent infection as a provirus, whose genome is integrated into the host dna, and does not replicate in cells in g0 phase . When the infected t lymphocytes are stimulated with antigen presentation from dcs, they proliferate triggering htlv-1 replication . During the replication stage, a viral protein, tax, activates nf-b and promotes the growth of infected cells via upregulation of il-2 and il-2 receptors [6, 7]. Nf-b also activates the long terminal repeat (ltr) of htlv-1 genome, which further enhances viral replication . The replicated virus induces a host immune response, and cells infected with virus are eliminated by the induction of cytotoxic t cells specific for htlv-1 tax . In patients with htlv-1-associated myelopathy / tropical spastic paraparesis (ham / tsp) this leads to the rapid elimination of infected cells through the induction of proinflammatory cytokines and cytotoxic t lymphocytes . So, in order to escape from acquired immune responses, htlv-1 needs minimum replication and latent infection . Some virus proteins are known as negative regulators of replication . The htlv-1 basic leucine - zipper factor (hbz) protein suppresses tax - mediated transcription activation of the viral ltr . P30 also contributes to the expression of tlr2, tlr4, and tlr9, and activation of irf4 . In monocytes, the tlr2 gene promoter is regulated by the transcription factors sp1, sp3, and pu.1, and the tlr4 gene by isre and pu.1 . P30 downregulates the expression of both tlr2 and tlr4, because it binds to pu.1 and prevents it from binding to dna . This leads to the suppression of dc maturation, and of their subsequent migration to the lymph nodes . Furthermore, p30 suppresses the enzymatic activity of glycogen synthetase kinase 3 (gsk3) through promotion of the phosphorylation of nine serine residues . This leads to the induction of il-10, which suppresses the function of macrophages, and also the maturation and activation of dcs . In fact, serum il-10 levels are elevated in patients with adult t lymphocyte leukemia . These immunosuppressive properties of il-10 indirectly contribute to the inhibition of virus replication and to the suppression of virus - induced immune responses . Activation of infected t lymphocytes by dcs in the lymph nodes and cell - to - cell transmission of virus are considered to be important for virus proliferation in human . The viral p12 protein suppresses cell surface expression of both mhc class i and the il-2 receptor, and also suppresses linker for activation of t cells (lat), which is an adaptor protein required for t cell activation . This results in suppression of t cells and dystunction of the stimulation / activation by dcs via the t cell receptor . Htlv-1 causes proliferation of infected cells rather than virus and suppression of host immune responses, which helps it to maintain a latent infection in order to survive . Hiv can infect cd4 t lymphocytes, monocytes, macrophages, and dcs, and preferentially replicates in activated t lymphocytes and activated macrophages . Hiv establishes a latent infection in resting t lymphocyte, and t lymphocyte activation by dcs or stimulation with il-2 is thought to trigger active replication of the virus . Apolipoprotein b mrna editing enzyme catalytic polypeptide - like 3 g (apobec3 g) is important for this resistance [16, 17]. The antiviral mechanisms of apbec3 g are considered to act via inhibition of viral reverse transcriptase (rt). It also produces a transition (g to a) in the dna strand transcribed by rt due to its cytidine deaminase activity . However, a mutant lacking cytidine deaminase retains antiviral activity . Low molecular weight complexes of apobec3 g (lmm: 70100 kda) have antiviral activity, but high molecular weight complexes (hmm: 700 kda) do not . The vif protein of hiv converts lmm to hmm, and promotes proteasome - dependent degradation of the complex, so hiv can counteract its antiviral activity . T cell activation leads to conversion of lmm to hmm (proviral environment). On the other hand, ifns induce lmm type (antiviral environment) [19, 20]. During the early stage of hiv infection, clinical symptoms show sings of immune system activation such as flu - like symptoms, rather than immunosuppression . Immunological activation is caused by rna40, an oligonucleotide derived from hiv, which activates pdcs, mdcs, t lymphocytes, and monocytes via tlr7 and tlr8 . The virus proteins, tat and vpr, induce proinflammatory cytokines via activation of nf-b [22, 23], which then enhances the transcription of the virus genome via the ltr . Il-8 also contributes to the propagation of the virus via the accumulation of t lymphocytes . So, hiv maintains a latent infection in order to survive within the host . The vpu protein suppresses nf-b activation by inhibition of proteasome - dependent degradation of ib . This causes suppression of tnf- production by macrophages via tlr4 signaling . Because tnf- is necessary for maturation and translocation of dcs (including langerhans cells) to the lymph nodes, downregulation of tnf- leads to suppression of acquired immune responses, and so prevents inhibition of virus proliferation by activated t lymphoctes . These strategies contribute to the suppression of hiv proliferation in the lymph nodes, and also inhibit the propagation of infection . Cosuppressive molecules, such as b7-h1 on dcs and pd1 on t lymphocytes, are upregulated in patients with hiv, and induce apoptosis through their interaction with dcs and t lymphocytes . Although, virus particles and virus proteins are found in the blood of hcv patients . Immune responses against hcv may be weak, but infected cells are attacked and eliminated . In order to survive in the host, hcv maintains a chronic infection by suppressing the host immune responses . Both hcv core and ns3 proteins activate nf-b and ap-1 via stimulation of tlr2, which requires tlr1 and tlr6 as costimulators, in monocytes and kupffer cells . This activation leads to the production of il-10 and tnf-, both found in hcv patients at a high titer . Il-10 suppresses the maturation of pdcs and the activation of t lymphocytes, and induces apoptosis in pdcs . The ns3-ns4a protein complex, which is a serine protease, degrades trif and ips-1/cardif / mavs / visa, which are essential for cellular signaling via tlr3, tlr4, and rig - i . This shutting off of trif- and ips-1-dependent signaling results in the suppression of ifn- and ifn- production, and in dysfunction of mdcs . This leads to the shutting off of tlr7 and tlr8 signaling, and to the suppression of maturation and differentiation of pdcs . On the other hand, nf5a suppresses traf2 dependent nf-b activation via interaction with nf5a and traf2, but suppress neither mek1 activation nor ikk-dependent nf-b activation . Infected dcs are thought to affect the production of tnf-, tnf- signal transduction, and chemotaxsis and maturation . Dysfunction of dcs, suppression of t lymphocyte activation, and a decrease in dc number due to apoptosis allow hcv to establish a chronic infection . It has also been reported that hcv - specific cytotoxic t lymphocytes share upregulated expression of the coinhibitory molecule pd-1, and that signaling from pd-1l (pd-1 ligand) on dcs results in suppression of hcv - specific cytotoxic t lymphocytes . Measles virus wild strains (clinical isolates) recognize cd150/slam (signaling lymphocyte activation molecule) as a receptor slam is strongly expresseed on memory t cells and b cells, but is also expressed on monocytes, t cells, b cells, and matured dcs . Infection of slam - negative mucosal epithelial cells with measles virus wild strains is thought to be mediated by an as yet unknown the ha protein of wild virus strain, but not the laboratory strain, induces cytokines such as il-1, il-1, il-6, il-8, and il-12 via the tlr2 signaling pathway . These cytokines activate and recruit immune cells to the site of inflammation, where the activated immune cells are infected with measles virus via slam . Interaction of the ha protein with slam suppresses tlr4-mediated il-12 production, but not il-6 and tnf- production . However, il-12 production mediated by other tlr signaling pathways (i.e., not via tlr4) is unaffected by the ha protein . These observations suggest that slam is a coupling factor for tlr4, and that the ha protein inhibits this function . The ha protein also interacts with a c - type lectin, dendritic cell - specific icam-3-grabbing nonintegrin (dc - sign) on dcs, and activates the serine / threonine protein kinase raf-1 via the ras signaling pathway . Dc - sign - mediated raf-1 activation induces phosphorylation of nf-bp65 on ser-276, and its subsequent acetylation . That virus proteins are synthesized de novo, the host cells, such as monocytes and dcs, infected with measles virus show suppressed il-12 production and tlr signaling, via tlr2, tlr4, tlr7, and tlr9 . Pdcs infected with the measles virus showed suppressed ifn production and dysfunction of maturation [39, 40]. Both monocytic cell lines u937 and thp-1 and human peripheral blood mononuclear cells infected with measles virus show markedly suppressed tlr2- and tlr4-mediated proinflammatory cytokine induction via nf-b and ap-1 . However, epithelial cells infected with measles virus show constitutive activation of nf-b and proinflammatory cytokine production, and these are further enhanced by treatment with tlr agonists such as lipopolysaccharide (lps). Monocytic cell lines infected with the mumps virus, which also belongs to the orthomyxoviridae family, show constitutive activation of nf-b and constitutively high levels of il-8 production . In monocytic cells infected with the measles virus, lps - induced ubiquitination (probably k63-linked type) of tnf receptor - associated factor 6 (traf6) is suppressed and does not form active complexes of tak1, tab2, and traf6 . An ubiquitin - modifying enzyme a20, which is a host nf-b negative regulator, is upregulated in measles virus - infected monocytic cells, but not in infected epithelial cells . The promoter region of the a20 gene shares two nf-b binding sites and a negative regulatory motif, elie, which is located upstream of, and adjacent to the two nf-b binding motifs . Measles virus p protein (phosphoprotein) interacts with the elie motif, and activates transcription of a20 . P protein is thought to release the suppressed a20 transcription machinery independently of activated nf-b . . However, the measles virus v protein, which is formed by rna editing of the p genome and has an n terminal amino acid sequence identical to that of p protein, does activate nf-b . The balance of the expression levels and time courses of the p and v proteins may also contribute to the cell - type specific suppression of tlr signaling pathways . The ns1 protein of type a influenza viruses suppresses innate immune system signaling activated by the pamps, via tlr3, tlr4, rig - i, and mda5 system . The mechanism of suppression is mainly via inhibition of irf-3 phosphorylation, leading to suppressed induction of ifn-/, and ifn-1, 2, and 3 . In addition, inhibition of nf-b and ap-1 activation in influenza virus infected cells leads to suppressed proinflammatory cytokine production, for example, il-8 and tnf-. Influenza virus ns1 protein is known to be a multifunctional protein able to inhibit the type i ifn induction, the ifn - induced antiviral activity, the binding and sequestration of dsrna, the interference with the host mrna processing, the facilitation of preferential viral mrna translation, and the inhibition of dc activation [44, 45]. Rsv f protein causes tlr4-mediated nf-b activation during the early infection stages of infection that is dependent upon virus replication . Il-1, il-6, and il-8 are induced via nf-b activated by the stimulation of tlr4 . During the late stages, the viral g protein is produced, and secreted, which then suppresses tlr4-mediated signal transduction . The cysteine - rich (gcrr) region of the g protein is also important for nf-b suppression . Following the interaction of virus proteins with host cell surface proteins, the viral m2 - 1 protein inhibits the translocation of rela, a component of nf-b, to the cell nucleus . Expression of the viral nonstructural proteins, ns1 and ns2 inhibit activation of irf3 induced by tlr3, tlr4, and rig - i signaling, and also suppress ifn production . However, ns1 and ns2 proteins have little effect on nf-b or ap-1 activation, so the production of proinflammatory cytokines may be effectively induced in the rsv - infected cells . Orthopox viruses, including the vaccinia virus, produce proteins that mimic cytokine receptors, such as those for ifn-, ifn-, ifn-, il-1, il-18, and tnf-, and disturb the cytokine signal transduction system . Other viral proteins also disturb the intracellular signal transduction systems . The viral a46r protein has a tir domain, and interacts with myd88 and trif, suppressing both il-1 and tlr signal transduction, but not tnf- signal transduction . The viral a52r protein binds to irak2, suppresses traf6-dependent ikk and nf-b activation, and then inhibits production of il-8 and rantes . However, a52r also binds to traf6 and promotes polyubiquitination of traf6, tak1 activation, mapkk6 phosphorylation, and activation of the jnk - p38 map kinase pathway . The viral n1l protein interacts with the ikk complex (ikk-ikk-ikk), tbk1, and ikk, and then suppresses the activation of nf-b and irf-3 . Vaccinia virus not only suppresses proinflammatory cytokines, but also induces production of an immunosuppressive cytokine il-10, which shifts the th1 response and suppresses cellular immunity . Human monocytes infected with vaccinia virus produce il-10, and this il-10 is then further upregulated by stimulation with lps . During the early stages of infection, the cytokines produced enhance both sensitivity to lps and the production of tnf-. Tnf- suppresses the formation and maturation of virus particles, and induces apoptosis of infected cells . The induction of tnf- is considered to be a host defense response . On the other hand, the receptor internalization and degradation (rid) complex, which consists two e3 products, e3(10.4 k)/rid and e3(14.5 k)/rid, suppresses cell surface expression of fas, tnf - related apoptosis - inducing ligand (trail) receptor 1, trail receptor 2, and tnf receptor 1 [54, 55]. This results in the shutting down of the tnf--mediated signaling pathways in the infected cells . Lps - induced mcp-1 and il-8 production are suppressed through the inhibition of nf-b and ap-1 activation . Rid does not alter the expression levels of tlr4, and so is thought to affect other components of the tlr signal transduction pathway . Hcmv modulates nf-b activity during the various stages of infection . During the early stages, nf-b then contributes to the induction of proinflammatory cytokines, to the expression of virus immediate early genes, and to the replication of the viral genome . The viral us28 protein, which is a hcmv - encoded chemokine receptor, constitutively activates both nf-b and phospholipase c signaling pathways . The activated nf-b mediates the upregulation of the host serine / threonine protein kinase, receptor - interacting protein - like interacting caspase - like apoptosis regulatory protein kinase (rick). Rick, which has a caspase - recruitment (card) domain, functions downstream of the pattern recognition receptors (which include the tlr, rlr, and nlr family members) and mediates nf-b and map kinase activation . Rick suppresses the replication of hcmv in cooperation with active nf-b and ifn- . Not to be outdone, the late gene products of hcmv also suppress nf-b activation [60, 61]. Il-6, il-8, and mcp are not induced in hcmv - infected cells and chemotaxsis, activation, and maturation are all suppressed . Ebv mainly infects b cells, although it can also infect t cells, nk cells, and epithelial cells . Both the attachment of ebv to receptor cd21 and the interaction of the virus glycoprotein gp350-gp250 with tlr2 activate nf-b . The virus gb and gh proteins are also candidates for tlr2 ligands . Immortalization of b cells by ebv infection is due to the activation of nf-b by the latent membrane protein 1 (lmp1), and antigen stimulation - like signaling of the b cell receptor by lmp-2 . Tlr2 signaling suppresses the transcription of the tlr9 gene via activation of nf-b containing p65 protein . So downregulation of tlr9 and upregulation of tlr7 and myd88 are observed in ebv - infected cells . Cell proliferation is thought to be driven by tlr7 signaling activated by virus rna, because the tlr7 antagonist irs661 suppresses cell division . Small rna encoded by the ebv genome (ebv - ebers) activates tlr7 signaling, and induces il-10 production . However, activated irf-5 is negatively regulated by ebv - induced irf-4 and a splice variant of irf-5 (v12irf-5). This suggests that the tlr7 signaling system play a role in the cell division of ebv - infected cells, and in the establishment of persistent infection . Lytic infection and production of virus particles are observed during the late stage of ebv infection . This leads to the downregulation of proinflammatory cytokines, the upregulation of tlr9, and the suppression of tlr7 function through interaction of tlr7 and tlr9 . Viruses modulate both the innate and acquired immune systems using a number of clever strategies . The goal appears to be to survive within the host for a long time, rather than efficient replication . Excessive replication would lead to detection and elimination by the host innate and acquired immune systems, thus bringing about the death of the virus . The virus needs to strike a balance between activation and suppression of host immune response . It is likely that each virus has developed various strategies to modulate the host immune response individually, and viruses that have succeeded in creating a good balance between host and parasite have survived . Modification of tlr signaling is a promising strategy for treatment of cancer, allergy, and infectious diseases [64, 65]. Especially, the immunomodulators should not generate resistant virus to drugs compared to antiviral drugs targeting viral proteins . As an existing example of virus infection, imiquimod, which is a tlr7 agonist, have been applied to an infectious disease caused by human papilloma virus, namely, condylomata acuminata . Several issues must be considered for the clinical application of tlr signaling . Among the greatest is assessment of what are keys of host defense or virus survival in view of a series of viral infection process . For example, human herpes simplex virus 1 (hsv-1) requires activated nf-b for its efficient replication . On the other hand, nf-b has a key role in inflammatory reactions via transcription activation of proinflammatory cytokines, cell adhesion molecules, and mhc . Thus, activation of nf-b is a double - edged sword for hsv-1 . In the host side, for example, intestinal epithelial cells express low levels of tlrs and high levels of negative regulators of tlr signaling, such as tollip [68, 69]. Also, in intestinal immunity, nf-b activation in a subset tlr signaling in dcs and macrophages is suppressed by a negative regulation ibns . The dysregulation of tlr signaling in the lumen of intestinal epithelial causes limit chronic inflammatory activation induced by commensal bacteria . Various tlr polymorphisms have been found, and some of them are shown to contribute to dysregulation of tlr signaling . The dysregulation has been suggested to be linked with a number of disease sensitivity and condition depending on individual differences [71, 72]. In conclusion, the molecular mechanisms involved in modulation of host immune systems, including tlr signaling, give us important hints on how to overcome infectious diseases caused by viruses.
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The increased life expectancy of dogs over recent years, probably due mainly to advances in canine nutrition and health care, has also been associated with an increased prevalence of cardiovascular disease . The vascular endothelium represents a widespread and interactive organ with various biological actions including barrier function, secretion of anti / prothrombotic factors, leukocyte and platelet adhesion and, very importantly, regulation of vascular tone [2, 3]. Macrovascular disease and heart failure are preceded and predicted by increased apoptosis of endothelial cells and dysfunction of the vascular endothelium including increased endothelial cell turnover to maintain an intact endothelial lining, increased smooth muscle cell migration due to an impaired endothelial barrier function, and a loss of vascular elasticity leading to an increased afterload [2, 46]. Moreover, it is well established that heart failure, experimentally induced in dogs by rapid pacing, as well as vascular endothelial dysfunction relate to reduced generation and/or exaggerated degradation of nitric oxide (no). Reduced bioavailability of no is associated with exaggerated superoxide anion production, increased oxidative stress, and sustained vasoconstriction [2, 8]. Such effects are believed to trigger vascular endothelial cell apoptosis and, in the long run, result in progression of human and canine valvular disease and heart failure [912]. Therefore, identification and characterization of vasoprotective agents and of their effects on endothelial cell function are of major importance in order to prevent or ameliorate the sequelae of endothelial dysfunction and vascular disease . So far l - carnitine and taurine, both known for their antioxidative capacity [13, 14], have been shown to be associated with beneficial effects in human endothelial cells as well in certain dog cardiomyopathies [1522]. Similarly, polyphenolic compounds such as isoflavones found in soybeans and tannins present in pomegranate extracts exhibit antioxidant and cyto- and cardioprotective activities [2330]. Concerning the human vascular endothelium, it has been proven by a considerable number of studies that factors often termed vasoprotective factors (no, shear stress, antioxidative agents) prevent, whereas atherosclerotic risk factors (oxidized / glycated ldl, hyperglycemia, proinflammatory cytokines, elevated free fatty acids) trigger apoptosis in cultured human vascular endothelial cells [3139]. Due to a lack of such models for the canine vascular endothelium, it was largely unknown to which extent the beneficial effects of l - carnitine, taurine, soy isoflavones, and pomegranate extract for the cardiovascular system relate to direct effects of those antioxidants on canine endothelial cell health and function . A previous recent study demonstrated that they could decrease the loss of viability of canine aortic endothelial cells (cnaoec) under conditions of oxidative stress . However the viability assay used in that study relied on total metabolic activity, and therefore it could be theorised that there is a risk of artificially good results if the tested substances concurrently promote significant excessive proliferation in surviving cells . Information is therefore still lacking regarding the impact of these substances on key indicators of functional health in canine endothelial cells such as apoptosis and proliferation . Moreover, it remains to be elucidated whether the observed effects of substances known to modulate these parameters are species dependent or can be extrapolated from species to species . Therefore, the present study aimed at development of an in vitro model on the basis of cnaoec, in order to characterize the potential beneficial or detrimental effects of different test agents with respect to canine endothelial cell proliferation and apoptosis . Different substances (gsh, nac, insulin sensitizers, etc . ), previously well characterized in human in vitro models for endothelial dysfunction [3438], were used as putative references for the development of our canine model and as internal controls for subsequent assays performed with test agents . We hypothesised that the reference substances would produce similar effects in cnaoec to those seen with human endothelial cells . We further hypothesised that the previously noted beneficial effect of the four test substances would be associated with stable or decreased apoptosis and proliferation, thus confirming the interest of these substances in developing a multidimensional dietary strategy to reduce the onset and progression of the canine endothelial degeneration involved in progressive valvular diseases . Cnaoecs and the respective media (cecbm, hbss) and growth supplements were purchased from cell applications, inc . Linoleic acid (lois), -linolenic acid (alens), fibronectin, bovine serum albumin (bsa), dimethyl sulfoxide (dmso), glutathione (gsh), and n - acetylcysteine (nac) were purchased from sigma chemical co. phosphate buffered saline (pbs) and trypsin ethylenediaminetetraacetic acid (trypsin edta) were from biowhittaker / lonza (belgium), human vascular endothelial growth factor (vegf) and basic fibroblast growth factor (bfgf) were purchased from biovision, and [methyl - h] thymidine from amersham pharmacia . Taurine and l - carnitine l - tartrate were from azelis pharma (paris, france), pomegranate extract (40% punicosides) from polinat (las palmas, spain), and soy extract (standardized at 40% soy isoflavones) from adm (decatur, il, usa). Gsh (10 mm, equivalent to 3.1 mg /ml), nac (5 mm, equivalent to 816 g / ml), vegf (25 ng /ml), bfgf (10 ng /ml), lois (50 m, equivalent to 14 g / ml), alens (50 m, equivalent to 13.9 g / ml), and the insulin sensitizers pioglitazone (pio) (50 m, equivalent to 17.8 g / ml) and rosiglitazone (rosi) (50 m, equivalent to 17.9 g / ml) were prepared and used as previously described [3438]. In brief, free fatty acids (lois and alens) were dissolved in ethanol, the insulin sensitizers (pio and rosi) were dissolved in dmso, all other reference substances were soluble in water . Where dmso or ethanol were required for the test or reference substances, the respective control cultures had the equivalent concentrations of dmso (1%) or ethanol (1.5%), respectively . L - carnitine, taurine, pomegranate extract, and soy extract were kept dry and protected from light (in brown flasks, covered with parafilm (bottle tops) and with aluminium foil). Before each experiment, fresh stock solutions were prepared for each substance: soy isoflavones were dissolved in dmso (250 mg / ml), all other test substances were soluble in water (50 mg / ml). Where dmso was required for the test substances, the respective control cultures had the equivalent concentrations of 1% dmso . The sample concentrations to be tested in the different assays, that is, 1, 50, and 250 g / ml, were estimated by reference to existing published studies using human endothelial cells or data reporting plasma concentrations in dogs or humans following oral supplementation [15, 19, 4244], and the absence of cytotoxicity of the proposed levels of these substances on cnaoec (data not shown). Experimental wells (with test or reference substances) were related to the respective control wells (without test or reference substances), set to 100% . Cnaoecs received as cryopreserved vial containing 500 000 cells were immediately frozen in liquid nitrogen and kept there until use . For further use, cells were thawed at 37c, resuspended in cecgm (according to the manufacturer's instructions), and cultured on fibronectin (0.0025% in pbs) coated (2 h/37c) cell culture dishes at 37c/5% co2 . For subcultures, confluent cells were rinsed with hbss and treated with trypsin / edta (6 ml/75 cm), followed by addition of 8 ml trypsin neutralisation solution (5% bsa in pbs). After centrifugation (5 min/220 g) the collected cells were replated in cecgm with a density of 5 000 to 10 000 cells / cm . The day after seeding and every other day, the medium was changed and confluency of the cells monitored by phase contrast microscopy . For proliferation and apoptosis assays, cells were used between passages 5 and 7 . Proliferation assays (see figure 1) were carried out as described previously [34, 35, 37]. In brief, confluent cnaoecs cultures were replated in flat bottomed 96-well tissue culture plates (10,000 cells per well) and allowed to adhere for 6 h . Subsequently, cells were exposed to h - thymidine (final concentration: 1 ci / ml = 37 kbq / ml) and the respective test agents for 48 h . After two washing steps with pbs, cells were trypsinized, lysed by a freeze thaw cycle, harvested, and incorporated . H - thymidine was counted in a tri - carb liquid scintillation analyser (canberra packard, meriden, usa). Results of experimental cultures exposed to the test substances are presented in relation to intraindividual control cultures (without test substances). Apoptosis assays (see figure 2) were performed as previously described [34, 35, 39]. In brief, semiconfluent plates (60 mm) of cnaoecs were labelled with h - thymidine (37 kbq / ml, 36 h) and were subsequently replated into 24-well culture plates (5 104 cells / well). After their exposure to the test substances (24 hours) cells were treated with lysis buffer (20 mmol / l tris.cl, ph 7.5, and 0.4% triton x-100 in pbs). Fragmented (apoptotic) radiolabeled dna in the supernatant was counted in a liquid scintillation analyser (canberra packard, meriden, ct) and was then related to total incorporated radioactivity of cells (quantified after digestion of the remaining suspension with 180 g / ml dnase, boehringer mannheim, germany). Results of experimental cultures exposed to test substances are presented in relation to intraindividual control cultures (without test substances). As depicted in figure 3, the antioxidant gsh, the insulin sensitizers pio and rosi, and the free fatty acids lois and alens significantly reduced proliferation in cnaoecs, such results being in line with previous work in different types of human vascular endothelial cells [35, 37]. Of note, however, vegf and bfgf, known to increase proliferation in human endothelial cells, did not provoke such proproliferative response in canine cells . Ethanol and dmso, used as solvents for ffas and insulin sensitizers, respectively, did not significantly affect cnaoecs proliferation . In cnaoecs, l - carnitine and taurine induced a slight but significant reduction of proliferation by 6.25% and 10.25%, respectively, at the highest concentration only (250 g / ml, see figure 4(a)). However, pomegranate - extract and soy isoflavones markedly reduced cnaoecs proliferation, even at a concentration of 50 g / ml, by 90% and 25%, respectively, and in a dose - dependent manner (figure 4(b)). Exposure of cnaoecs to 250 g / ml of these two substances led to an impressive and highly significant inhibition of the cells' proliferation to 3.75% and 15.5% of control (set to 100%), whereas pio and gsh reduced proliferation only to 62.25% and 54.75% of control, respectively (figure 4(b)). As shown in figure 5(a), the antioxidant gsh at 10 mm markedly reduced apoptosis (by 35%) in cnaoecs . In contrast, in cnaoecs exposed to 300 mol / l lois and alens, death rates dramatically increased, more than 55% of cells having undergone apoptosis after 48 h of incubation (data not shown). Such antiapoptotic effects of antioxidants as well as the proapoptotic response exerted by free fatty acids have previously been observed in human vascular endothelial cells [34, 35]. L - carnitine and taurine did not markedly affect apoptosis in cnaoecs, the statistical significance observed for 50 g / ml carnitine being probably related to the very low standard deviation observed for that particular concentration (figure 5(a)). The antiapoptotic effect of soy extract was significant only for the highest concentration tested (250 g / ml). In contrast, pomegranate extracts significantly reduced apoptosis at all concentrations tested and in a dose dependent fashion, showing the most striking effect at 250 g / ml by a reduction of 73% compared to control cells (figure 5(b)). Due to the increasing amount of evidence linking endothelial dysfunction to the onset and progression of heart failure in dogs [11, 12], it is important to have a method of screening and/or testing substances that may have potential to protect endothelial function in vitro . This is already widely done with human endothelial cells, and the markers of excessive proliferation and apoptosis are crucial indicators of dysfunction . However, until now, no such model existed using canine endothelial cells . This is why we began the study with the establishment of an in vitro cell culture model which allowed the reproducible analysis of these markers in response to different stimuli . This then enabled, as a second step, the identification of pro- and antiproliferative as well as of pro- and antiapoptotic agents . The reference substances were chosen on the basis of their abilities to exert anti- as well as proproliferative and anti- and proapoptotic responses in human endothelial cell culture models . Of note, the observed antiapoptotic and antiproliferative effects were similar in human endothelial cells and the tested canine endothelial cells for the antioxidants gsh and nac as well as for the insulin sensitizers pio and rosi [34, 35, 37]. The free fatty acids exhibit marked proapoptotic action in canine endothelial cells, which clearly exceeds the effects exerted in human vascular endothelial cells . Of course it must be noted that this relates to use of these particular free fatty acids in isolation, as opposed to the more normal dietary situation where a balance of omega-3 and omega-6 oils will be present, and where the ratio of these oils has already been shown to be important . In contrast to human umbilical vein endothelial cells, vegf and bfgf, were not able to provoke a proproliferative response in cnaoecs . In that context it is of note that it has previously been speculated that the effects of natural antioxidant molecules in endothelial cells can be species dependent . As could be observed for the reference substances gsh and pio, which were used as internal controls in each assay performed for the test substances, the model exhibited considerable reproducibility throughout the whole study, even if different settings (coating, growth factor supplement, etc .) Were employed (data not shown). This therefore partially confirms our initial hypothesis that substances shown to be beneficial for human endothelial cells will also produce beneficial results in cnaoecs, but intriguingly suggests that it is not possible to extrapolate these results directly across species due to occasional differences in the intensity of the effects . For the first time this study shows that extracts of pomegranate and soy isoflavones are able to exhibit dramatic antiapoptotic activities in cnaoecs . Pomegranate extract's antiapoptotic effect was already detectable at the lowest concentration tested (1 g / ml) and was dose - dependently sustained until the highest concentration applied (250 g / ml). Accelerated apoptosis of vascular endothelial cells is relevant in the development and progression of cardiovascular disease [46]. We have previously shown that vasoprotective agents such as antioxidants (including lipoic acid and gsh) or insulin sensitizers exhibit both antiapoptotic and antiproliferative effects in human vascular endothelial cells . Those effects could prevent loss of the endothelial barrier function (due to accelerated endothelial cell apoptosis) and exhaustion of the endothelial cells' proliferative capacity (due to accelerated proliferation). Both the antiapoptotic and antiproliferative activity of pomegranate and isoflavones in cnaoecs therefore suggest a vasoprotective action of those polyphenolic compounds in dogs, which could beneficially affect chronic mitral valvular insufficiency (cmvi), the major cause of heart failure in this species . Since progression of cmvi is assumed to be triggered by endothelial dysfunction and the latter vice versa is potentiated by cmvi - associated increased cellular oxygen demand [4, 11, 12, 17, 4648] the cytoprotective antiapoptotic as well as antioxidative effects of pomegranate and soy isoflavone extracts could beneficially affect the progression of cmvi to heart failure in dogs . Both of these agents are complex biological substances containing multiple potentially active polyphenols or isoflavones . It was not possible or appropriate to attempt to identify the balance of the effects of individual components of complex natural substances such as these, but indeed this kind of complexity using agents with a multi - faceted action has been proposed as a necessary and important part of rational use of antioxidants to prevent cardiovascular disease . L - carnitine and taurine were previously shown to exert protective effects against oxidative stress in human endothelial cells [15, 22]. In a recent study we have shown that pomegranate extract, alone and in combination with soy isoflavones, taurine, and l - carnitine, also shows strong protective effects against oxidative stress in cnaoecs . In that context it is of note that the survival signaling pathways of the bcl-2 family, pi3kinase, and estrogen receptor are assumed to mediate the protective antioxidative effects of the soy - derived isoflavones genistein and daidzein . Such pathways have previously been shown to mediate antiapoptotic effects described for lipoic acid in human vascular endothelial cells . Since taurine and l - carnitine, in contrast to extracts of pomegranate and soy isoflavones, did not affect apoptosis in canine aortic endothelial cells, it is tempting to speculate that different mechanisms are involved in those substances' antioxidative and beneficial vascular effects in dogs . It is also worth noting that any results obtained for extracts of pomegranate are probably specific to each type of extract . It has been shown that extracts obtained from different parts of the plant have widely varying compositions of active components [49, 51]. Concerning the concentrations applied, there is a wide range of concentrations described in the literature for antioxidants, depending on species and compound . To cover such variety the test substances were used in a broad range from 1 to 250 g / ml . The lack of any cytotoxic effects at the highest concentrations we used also provides some reassurance regarding the potential use of these compounds . One limitation of a study such as this is related to the use of an in vitro cell - based model . Indeed it is possible that there may be differences in the responses of cells in the in vivo situation compared to the in vitro environment, and it is also not possible to assess completely the impact of the diseased state on the function of the endothelial cells . One possible solution could be to use isolated segments of blood vessel to assess vasorelaxation responses . However this equally would have significant limitations and would preclude the direct assessment of key markers such as apoptosis . On balance, as substances having beneficial effects on proliferation and apoptosis of human endothelial cells in the in vitro situation have been shown to also provide in vivo benefits, this type of study is a rational step providing key information regarding the potential benefits of these substances in the canine species . In conclusion, this is the first study showing that pomegranate extract and soy isoflavones inhibit apoptosis and proliferation in canine aortic endothelial cells . This supports our hypothesis that these agents could have a role in prevention or amelioration of clinical endothelial dysfunction and therefore progression of cardiovascular disease in dogs [47, 48]. Further studies will, however, be necessary to evaluate the underlying mechanisms and to which extent the antiapoptotic and antiproliferative effects observed in our canine endothelial cell culture model could also apply to the in vivo situation.
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A young boy, 16 years old, presented with complaints of poor vision in the left eye since childhood ., he had a vision of 20/200 in the right eye (re) and 20/20 in the left eye (le). Zonules were lacking around the fissure between the two lobes [fig . 1]. 2] revealed a bilobed lens in the anterior segment of the eye with normal posterior segment . Partial congenital aphakia is a rare condition in which the lens appears to be partially divided by a deep furrow.1 in the case of congenital duplication of lens reported by thakkar et al.2 two lenses were placed obliquely in a straight line with a clear area in between . Richardson3 reported a case in which the two lenses were asymmetrical, one being larger than the other . The mechanism for this anomaly is not known yet . Whether it is part of a phylentogenic spectra or represents development from two embryonically separate lens remains a question . Duke elder has tried to explain the occurrence of the two entities based on separate embryonic developmental sequences.1 he suggested that the metaplastic changes in the surface ectoderm leads the lens plate to invaginate at two place and forms two lens vesicles which results in double lens.1 this suggests that the lens is divided before the lens starts developing . Our case has a deep furrow in the center fig . 1 with localized absence of zonules . We, therefore, suggest that once the deep furrow is formed due to absence of zonules, it may go further (deep) enough to divide the lens into two distinct parts . Therefore, the absence of zonules is significant and these furrows may be deep enough and progress to duplication . It is a possibility that the lens duplication might be occurring when the lens vesicle is single and it is the laxity and abnormal stretching which causes the deep furrow and possibly duplication . It is interesting to note that both the cases previously reported2 - 3 had lens place in a straight axis at opposite ends . This gives further credence to our theory of the fissure dividing the lens into two and the remaining zonules pulling them away from each other . Since lens coloboma occurs frequently such deep furrows (bilobed lens) are uncommon . We believe that it represents the missing link between the colobomatous lens and duplicated lens . We understand that a single case report may not be suggestive of any mechanism but nevertheless it does indicate a possible mechanism of lens duplication.
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The increasing prevalence of obesity represents a major public health concern in many countries, and healthy eating can play a predominant role in prevention strategies . In this context, it is important to identify effective nutritional strategies to help individuals achieve or maintain a healthy weight by making better food choices . One of these strategies consists in the regulation of food labeling, which includes the use of nutrition claims intended to provide a quick and easy way to identify foods with nutritional features of interest [24]. In this regard, satiating properties of foods induced by specific nutrients such as fibres or proteins are of particular interest [5, 6]. Indeed, there is convincing evidence that appetite sensations are important determinants of long - term energy intake [7, 8] and that satiety - enhancing foods may provide benefit to consumers by facilitating appetite control and compliance with weight - management efforts . Furthermore, the regulation of nutrition claims related to satiety is currently evaluated and developed by health organizations, which clearly recommend that such claims are supported by strong scientific evidence [10, 11]. While nutritional information related to the satiating effects of foods might be relevant for weight control, less is known about consumers' perception and understanding of such nutrition claims and how these claims can affect perceived appetite sensations . Indeed, nutritional information conveyed by food labeling can be difficult to understand for many consumers, particularly for older adults, adolescents, or those with less education [12, 13]. Nutrition claims can also be misunderstood, which may lead to misleading inferences or unexpected eating behaviors . For example, a recent study showed that a low - fat - labeled candy was perceived healthier than a regular - labeled candy, independent of caloric information . Healthy was considered less fattening and that the amount eaten was 35% higher than when the snack was described as unhealthy . With regard to appetite sensations, it has been demonstrated that a food considered as unhealthy is perceived to contribute more to weight gain and satiation, while imposed healthy eating can increase the sensation of hunger . Beliefs and expectations about the satiating power of a food can also affect perceived fullness, and this effect can persist as long as three hours after the meal . Another study showed that a meal presented as highly satiating, without having physiological satiating properties, can increase satiety potential more than a meal having physiological satiating properties, but presented as regular . This suggests that the psychological effect of a satiety - related claim can be as effective as a physiological effect related to intrinsic satiety properties and highlights the need to better understand this possible psychological effect . Individual factors such as sex, weight, or restrained eating could also influence the perception of foods and modulate behavioral change . As the perception of food can influence appetite sensations, it could be hypothesized that individual factors can also modulate the perception of appetite sensations . Since women generally consider themselves as being more knowledgeable about nutrition and having better eating habits and tend to pay more attention to nutritional information, it could be suggested that women would be more readily influenced by nutrition claims . Overweight / obese individuals and restrained eaters might also be more sensitive to nutrition claims related to healthy eating or weight loss since they can use it as a tool for weight management . Interestingly, one study showed that food products carrying satiety - related claims were generally not seen as a magic bullet to weight loss, but that restrained eaters were more prone to overinterpretation of these claims, that is to interpret it as directly delivering weight loss . Individual factors can thus affect perception of foods and perceived appetite sensations, but they can also act in a physiological way . Indeed, some studies have associated higher levels of restraint with an orexigenic hormonal profile [24, 25], suggesting that restrained individuals could more easily feel hunger . It has also been proposed that overweight / obese individuals are less connected to their internal signals than normal - weight individuals, making the detection of hunger and satiation more complex . Sex, weight, and restrained eating are thus variables of interest when studying appetite sensations, alone or in combination with nutrition claims . In summary, current literature suggests that nutrition claims may influence perception of foods and appetite sensations and that this influence could be modulated by individual factors such as sex, weight, or restrained eating . In that context, there is an urgent need to better understand the way nutrition claims should be used, taking into account possible side effects, and evaluate the relevance of some claims in a public health nutrition context . This study addresses this issue by evaluating combined and separated effects of nutrition claims and individual factors on appetite sensations . The main purpose of this study was to evaluate the impact of nutrition claims and individual factors on perceived appetite sensations . The specific objectives were (1) to evaluate the effects of healthy, diet, or claims on appetite sensations, (2) to evaluate if sex, weight, or restrained eating influence appetite sensations, and (3) to investigate if sex, weight, and restrained eating can modulate the effects of nutrition claims on appetite sensations . Note that the hedonic condition refers to ingredients generally considered as highly palatable and that the diet condition refers to satiating properties (see section 2.1 for a complete description). Our corresponding hypotheses are that (1) the snack in the healthy condition will be less satiating than in the diet or hedonic conditions, (2) individual factors such as sex, weight, and restrained eating will significantly influence appetite sensations, and (3) the satiating effects of the diet and hedonic conditions will be more important in overweight / obese restrained women . The study was conducted among 164 men and 188 women (aged 1865) from quebec city, canada, who were invited to taste and rate an oatmeal - raisin snack in a blinded and ad libitum context . Exclusion criteria were personal condition or history of diseases that could affect food intake (unstable weight in the last two months, pregnancy, breastfeeding, eating disorders, type 1 or 2 diabetes, and uncontrolled hypo- or hyperthyroidism), use of medication that might interfere with appetite or food taste (antidepressants, antipsychotic, or corticosteroids), and food allergies for safety reasons . Participants were randomly assigned to an experimental condition in a three (healthy versus diet versus hedonic) by two (restrained or not restrained) by two (normal - weight or overweight / obese) by two (men versus women) factorial design, which means that the recruitment was based on these characteristics in order to obtain a balanced number of participants in each subgroup . This design results in eight subgroups based on individual characteristics combined with the three experimental conditions tested, which leads to a total of 24 combinations for the analysis . The factorial distribution of participants is presented in table 1 . Initial randomization was performed according to self - reported weight and height and restraint score, but participants were reassigned according to their measured data if needed . Condition emphasized the favorable nutritional characteristics of the snack: the snack product that you have to taste today is a new high - fiber oatmeal snack made with healthy ingredients . You have certainly heard that whole oatmeal is good for your health because it contains soluble fibers . So, this new oatmeal snack is high in soluble fibers, as well as low in saturated fat and free from trans - fat . The diet condition focused more on beneficial satiating properties for weight management: the food product that you have to taste today is a new healthy high - fiber snack . You must have probably heard that whole - grain foods, like those that contain oat soluble fibers, are composed of complex carbohydrates which are digested slowly and which can delay the appearance of hunger . This new healthy snack has been especially designed to satiate, because it is a source of fibers, which make it an interesting choice for any persons concerned to reach and to maintain a healthy weight . Finally, condition underlined the sweet and pleasant taste of the snack using hedonic food words: (these are) new gourmet cookies made with fresh butter and old - fashioned brown sugar . So, these new cookies are a great treat with a pleasant, sweet taste . Cookie was never mentioned in the two first conditions, and that we specifically chose to use an oatmeal - raisin rather than a chocolate snack in order to facilitate the blinded context of the experiment, as oatmeal and raisins can be considered as healthy ingredients or not depending on the food containing it . Indeed, this psychological manipulation was previously shown to be effective in changing the healthiness perception of food [16, 28]. In spite of the description, the study was approved by the institutional review board of universit laval (#2009 - 117/26 - 05 - 2009) and was registered in the clinical-trials.gov registry (nct01141140). Participants came to the institute of nutrition and functional foods (universit laval) between 8:30 a.m. and 7 p.m. at a single occasion . They had to be in a fasting state for at least two hours prior to their visit . They were given a food description according to the experimental condition they were assigned and then had 10 minutes to taste the snack ad libitum in a private room . Participants stayed for approximately two hours after the taste - rating task to complete appetite sensations scales, questionnaires, and anthropometric measurements . They were told the real goal of the study only at the end of the visit and were free to withdraw their participation at that moment . Appetite sensations are defined as follows: hunger refers to the physiological recognition of a need to eat, while desire to eat reflects the motivation to eat, fullness is a physical feeling that could be related to the degree of stomach filling, and satiety refers to the processes that inhibit further intake after an eating occasion has ended . Appetite sensations were evaluated with unipolar 150 mm visual analog scales (vas) (adapted from hill and blundell). These scales are anchored at the two ends with the extremes of the subjective feeling evaluated . Vas have been widely used in the past to evaluate appetite sensations and are considered reliable for single meal protocols [31, 33]. Participants rated their appetite sensations before the test meal (time = 10) as well as immediately after (time = 0) and 20, 40, and 60 minutes later . The first measure (time = 10) was taken immediately before the description of the snack . Four appetite sensations were measured: desire to eat (how strong is your desire to eat? ; very low desire to very high desire), hunger (how hungry do you feel? ; not at all hungry to extremely hungry), fullness (how full do you feel? ; not at all full to extremely full), and prospective food consumption (pfc) (how much food do you think you could eat? ; none at all to a large amount). The vas values were calculated by measuring the distance in mm with a ruler from the left end of the scale to the mark drawn by the participant with a precision of 0.5 mm . One - hour area under the curve (auc) was calculated from time zero to time 60 with the trapezoid method . As recommended by blundell et al . The auc allows an overall view of the appetite sensation response, while the raw vas data allows comparisons at each time . Appreciation of the snack (in general, how much do you appreciate the food you just tasted?) Was evaluated with a unipolar 150 mm vas labeled from bottom to top with not at all, moderately, and enormously . A validated food frequency questionnaire (ffq) the restraint scale allowed the distinction between restrained eaters (score 12 for men and 15 for women) and unrestrained eaters (score <12 for men and <15 for women). The validity of the restraint scale for the measurement of restrained eating has been previously reported [3739]. The food pleasure scale (fps) was also used to evaluate the pleasure associated with eating . Body mass index (bmi) was calculated from weight and height measured after the completion of all questionnaires . Participants were classified as normal - weight if they had a bmi under or equal to 25 kg / m and as overweight / obese if they had a bmi higher than 25 kg / m . Energy needs were estimated from the mean of ffq data and the harris - benedict formula, as previously performed in our research institute . With a mean cohen's d estimate of 0.35, power analyses for anovas testing main effects and interactions indicated that a sample size of 180 males and 180 females would allow the detection of significant differences with an alpha level of 0.02 and a power (1 error probability) of 0.90 . One - way analyses of variance (anovas) were used to assess differences in initial characteristics between experimental conditions . In line with the factorial design of the study, factorial repeated measures anovas were performed with mixed procedures for the analysis of the appetite vas (desire to eat, hunger, fullness, and pfc), with time, experimental condition, sex, bmi, and restrained eating used as independent variables and tested in interactions . Age and initial appetite sensations (time = 10) were included in the models as potential confounding factors, as they may affect appetite sensations [29, 44]. Adjustments for energy needs and total amount eaten were also made to palliate to the fact that the amount eaten was ad libitum and therefore not adjusted for energy requirements (as the study also aimed to measure the effect of claims on food intake). Time of the day was also added as a covariate in case the two hours fasting period was not long enough to standardize initial appetite sensations . Finally, appreciation of the snack and pleasure associated with eating were added as covariates as hedonic eating indicators . Similar procedures without repeated measures were used for the analysis of auc for all appetite sensations . The slice option was used to investigate significant experimental conditions effects in each of the eight predetermined subgroups (based on sex, weight, and restrained eating). The structure of covariance matrix was taken into consideration for all statistical models to ensure an optimal fit to the data . A p value <0.05 was considered statistically significant for all tests, with bonferroni corrections made for all multiple comparisons . All analyses were performed with statistical analysis software version 9.3 (sas institute, cary, nc, usa). A total of 352 participants were recruited and completed the study . From these participants, 11 (4 men and 7 women) were excluded from the analysis because of missing values in covariates . The remaining total of 341 participants (160 men and 181 women) were thus included in the present study . One missing value was also observed for the initial pfc sensation (time = 10), which led to the exclusion of one participant from the pfc analysis because of the necessity of this covariate in the statistical model . No significant differences were found between conditions for any initial characteristic . As explained previously in section 2.6, all the following results are adjusted for age, time of the day, energy needs, amount eaten, and initial appetite sensations . Note that only initial appetite sensations and total amount eaten contributed significantly to the models . No main experimental condition effect was observed for any of the four appetite sensations evaluated, when expressed in both vas means (f(2,306) = 1.14 for desire to eat, f(2,306) = 1.54 for hunger, f(2,306) = 0.66 for fullness, and f(2,305) = 0.68 for pfc; p = 1.00) and auc (f(2,306) = 2.99 for desire to eat, f(2,306) = 3.20 for hunger, f(2,306) = 1.06 for fullness, and f(2,301) = 0.4440 for pfc; p = 1.00). Mean values for all vas at each time point (10, 0, 20, 40, and 60 minutes) as well as their related auc are presented in table 3 for each experimental condition . Appetite sensations were affected neither by bmi (p = 1.00 for all vas values and auc), nor by restrained eating (p> 0.96 and p> 0.33 for all vas values and auc, resp . ). However, a main effect of sex was noted for all appetite sensations, with men reporting higher vas values than women for desire to eat, hunger, and pfc (p <0.004) and lower vas values than women for fullness (p = 0.008). As presented in table 4, cohen's d effect sizes (ess) ranged from 0.08 to 0.52, which represents small to moderate effects . An effect size comprised between 0.2 and 0.49 represents a small effect size, between 0.5 and 0.79, a moderate effect size, and 0.8 a large effect size . No sex differences were observed for desire to eat and hunger at baseline, but fullness was higher and pfc lower in women in comparison to men . Note that men had significantly higher energy needs and ate significantly more than women (data not shown). As previously explained in section 2.6, adjustments were made for these covariates for all results presented . Significant effects of nutrition claims on appetite sensations were observed in specific subgroups (based on combinations of individual factors). Among normal - weight unrestrained women, lower auc values for desire to eat (es = 0.76), hunger (es = 1.03), and pfc (es = 0.72) were observed in the diet condition compared with the healthy condition (table 5). However, significant differences between these two conditions were not observed at each time point . Some differences between diet and hedonic conditions and also between healthy and hedonic conditions were also noted among normal - weight unrestrained women . In fact, hunger was lower in the diet condition than in the hedonic condition at time 0 and was higher in the healthy condition than in the hedonic condition at time 20 (table 5). Among overweight / obese unrestrained men, a higher auc value for fullness was observed in the diet condition than in the healthy and hedonic conditions (ess of 1.19 and 0.99, resp .) (table 6). No significant differences were found between other subgroups for any of the four appetite sensations . The aim of this study was to evaluate the impact of food labeling and individual factors on perceived appetite sensations and more precisely to (1) evaluate the effects of healthy, diet, or claims on appetite sensations, (2) evaluate if sex, weight, or restrained eating influence appetite sensations, and (3) investigate if sex, weight, and restrained eating can modulate the effects of nutrition claims on appetite sensations . Associated results are that (1) claims did not affect appetite sensations, (2) sex had a significant influence on appetite sensations, but not weight and restrained eating, and (3) a significant interaction between claims and individual factors has been observed in normal - weight unrestrained women and overweight / obese unrestrained men . As previously reported, the psychological manipulation was effective in changing the healthy perception of the snack, as the healthy condition was considered to be healthier than the diet condition, itself considered healthier than the contrary to our first hypothesis, the three experimental conditions resulted in similar perceived appetite sensations, suggesting that a snack labeled with healthy characteristics is not necessarily perceived as less satiating than a snack labeled with satiating or hedonic properties . This finding is in contradiction with finkelstein and fishbach who demonstrated that participants felt hungrier after eating a healthy chocolate - raspberry bar than after eating the same bar described as yummy . However, few details are given in this study about the composition of experimental groups, and initial hunger sensation was not measured and then not used as a covariate, as performed in the present study . As our statistical models showed that initial sensations are important covariates to consider in the evaluation of appetite sensations, differences in statistical adjustments between studies may explain the different results obtained . Our finding is also in contradiction with arguin et al . Who demonstrated that highlighting the satiating properties of a meal increases satiety potential against a control condition . The different types of food (i.e., snack versus meal) used and the absence of a control condition (i.e., without any description) in our study may explain why we did not observe such a difference . It could also be explained by the fact that our diet condition was not only targeting satiating properties, but also weight control . As we did not measure the expected satiating power (as performed previously for perceived healthiness) in any case, the absence of an effect of nutrition claims on appetite sensations in our study implies neither a contraindication nor incentive to their use in a public health context . As hypothesized, sex had a significant influence on the perception of appetite sensations, regardless of nutrition claims, energy needs, and amount eaten (men had significantly higher energy needs and ate significantly more than women). Indeed, men reported higher levels of desire to eat, hunger, and pfc and lower levels of fullness . These results suggest that women could feel more easily satiated than men and could be less tempted to prospectively eat more food . Some studies have already reported similar findings [7, 44], while other studies found no sex differences or mixed results depending on the appetite sensation observed and experimental conditions (ad libitum versus controlled). The fact that baseline fullness and pfc were, respectively, higher and lower in women than in men is also of interest . It suggests that these two sensations could differ between men and women even before food consumption . Sex differences in the perception of appetite sensations could be explained by differences in hormonal profiles . These observations involve that public health nutrition interventions may not have the same effects in men and women and that individual or group interventions should be adapted in consequence . According to our findings, women could be more responsive to an intuitive or mindful eating approach than men, that is, to eat in response to physical sensations such as hunger and satiety signals [4951]. Health at every size (haes) programs, endorsed by the association for size diversity and health, are an example of interventions addressing the recognition of appetite sensations . Other programs are also sex - specific, focusing more on body image for women and energy and body functions for men . Current literature shows that obese individuals have lower fasting ghrelin levels and that their ghrelin response to food intake is less pronounced than in normal - weight individuals . This is in line with the proposition that overweight or obese individuals could be less sensitive to their internal appetite sensations . However, we found no difference between normal - weight and overweight / obese individuals, which has been found in past literature . Another study proposes that hormonal response to food intake could be dependent of a bmi by sex interaction, suggesting that the study of bmi alone is not sufficient . Appetite - regulating hormones are not necessarily related to subjective appetite sensations, which could explain why we obtained different results as we only measured perceived appetite sensations and not appetite - regulating hormones . Also, we did not differentiate between overweight and obese individuals, our sample including more overweight (n = 124) than obese (n = 51) individuals . No main effect of restraint status on appetite sensations was observed . This was unexpected as restrained eating has been previously associated with higher levels of perceived hunger . It has also been already associated with higher levels of ghrelin [24, 57]. Hooper et al . Also demonstrated that a high degree of weight cycling was associated with an appetite - stimulating hormonal profile . As mentioned previously, however, appetite - regulating hormones are not necessarily well related to subjective appetite sensations . On the other hand, other studies found no effect of restrained eating on hunger [44, 55]. Restrained eating can be measured either by the restraint scale (as performed in the present study) or by the restraint scales included in the three - factor eating questionnaire (tfeq) or the dutch eating behavior questionnaire (debq). The restraint scale combines the measure of concern with dieting and weight fluctuation, whereas both tfeq and debq measure attempt to restrict eating, independent of weight fluctuation . The choice of the questionnaire used to classify participants as restrained or not could then explain differences in results obtained between studies . As proposed earlier, it is also possible that restrained eating needs to be considered in interaction with sex or bmi to reveal significant effects . For example, martins et al . Showed that restrained eating in women could be associated with lower levels of hunger and higher levels of fullness . Even if weight status and restrained eating are important factors to consider in individual consultation, our results suggest that they do not necessarily need to be taken into account for the evaluation of appetite sensations . To our knowledge, this is the first study to explore the influence of nutrition claims on appetite sensations according to a combination of individual factors, namely, sex, weight, and restrained eating . Our hypothesis that overweight / obese restrained women would be more easily satiated in the diet and hedonic conditions in comparison with the healthy condition was not confirmed as a satiating effect of the diet condition was rather observed in normal - weight unrestrained women and overweight / obese unrestrained men, while no satiating effect of the hedonic condition was noted . Being more easily influenced by satiety - related claims could either indicate a greater susceptibility to external factors, or in contrary a better predisposition for the recognition of internal satiety signals . An effect of nutrition claims was only noticed in individuals presenting a low level of cognitive restraint, which has already been associated with a lower susceptibility to external influences [20, 56]. It is then more likely that the diet this observation highlights the relevance of considering individual factors in interaction when studying appetite sensations . Our results also suggest that nutrition claims emphasizing satiating properties could be more effective in reducing perceived hunger than claims focusing on hedonic properties . More precisely, it suggests a potential benefit of satiety - related claims, at least in certain subgroups of individuals . First, it might be argued that the healthy and diet labels were not congruent with the food tasted, which could explain the absence of a main effect on appetite sensations . However, this argument is not supported as the manipulation effectively changed the perception of food . On the other hand, no control condition (i.e., without any description) was used, and the diet condition was not only targeting satiating properties but also weight management . Also, the amount eaten was not standardized, which however allowed participants to eat according to their physiological and psychological needs . The use of many multiple comparisons and bonferroni corrections could have led to a lack of power, but it can also be argued that the probability of committing type 1 errors is really small . This study also has strengths, such as a high number of participants recruited on the basis of a factorial design . This is also the first study to evaluate the role played by specific combinations of individual factors in the study of the effect of nutrition claims on appetite sensations . We investigated the impact of nutrition claims and individual factors on the perception of appetite sensations . Women felt more satiated then men regardless of nutrition claims, while bmi and restrained eating alone did not influence appetite sensations . No adverse effect of nutrition claims on appetite sensations was observed, which suggests no contraindication to their use . Positive effects of satiety - related claims have been observed only in specific subgroups of individual . In a real - world setting, foods labeled with satiety - related claims would possess demonstrated physiological satiating properties and therefore would probably induce a physiological effect (as opposed to a psychological effect in this study). As enhanced satiety may benefit dietary control or weight - management, it may thus be useful to inform consumers about these specific properties . To conclude, nutrition claims do not generally affect appetite sensations and the evaluation of their relevance sex is an important individual factor to consider when studying appetite sensations, while weight and restrained eating should be considered in interaction for a better understanding of their effects . More studies are needed to assess a significant and beneficial effect of nutrition claims on appetite sensations and eating behavior, and to better understand the contribution of individual factors.
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The course is divided into 5 modules, where the leading experts in the field provide online lectures within their area of expertise (table i). During the first module of the course therefore, topics that are covered during the introduction week are the nomenclature for the different subpopulations of evs as well as an introduction to the diversity of organisms releasing evs and the tissues and body fluids where evs can be found . Furthermore, one of the pioneers, professor emeritus philip stahl, shares the story about how he and his colleagues discovered exosomes in the early 1980s (4). The second module focuses on the biogenesis and release of evs and how this differs between the ev subpopulations: exosomes and microvesicles . Additionally, the different uptake mechanisms of evs when they are encountered by a recipient cell are covered in depth (10). As evs have been shown to contain functional rnas, proteins and lipids, this module also covers the different types of molecules present in evs as well as a brief overview on what the potential functions of these molecules are . Furthermore, the techniques that are commonly used to detect these molecules and to analyse the cargo of evs will be highlighted . In the third module, the focus is on the collection and processing of cell culture media and body fluids prior to isolation of evs . Here, considerations and guidelines that are important during the collection of the ev - containing material and when isolating evs from these fluids are discussed (11,12). This module will help the students to reflect over the many different choices, such as anticoagulants, collection time points and protein inhibitors, which are important for the outcome when working with a particular body fluid compared with conditioned media or other body fluids . This module also illustrates some examples of studies on evs from body fluids such as blood, urine, breast milk and cerebrospinal fluid and why it is of interest to analyse evs from these bodily fluids . The basic concepts and some guidelines for methods such as differential ultracentrifugation, density gradient centrifugation, size - exclusion chromatography and kit - based precipitation are covered . Furthermore, this module covers how the techniques are used in the field of evs as well as their limitations and benefits . The importance of evaluating the heterogeneity, purity and characteristics of the isolated vesicles regardless of isolation method is also highlighted (13). The fifth module covers some of the different techniques that can be used to characterise evs . Here, the basic concepts for techniques such as electron microscopy, cryo - transmission electron microscopy (cryo - tem), flow cytometry, atomic - force microscopy (afm) and nanoparticle - tracking analysis (nta) are covered . Furthermore, this module covers how the techniques are used in the field of evs as well as their limitations and benefits . This course is recommended for anyone interested in the field of evs including biology and medical students and phd students without previous experience in the field as well as clinicians, cell and molecular biologists and researchers who want to broaden their understanding of the field and deepen their knowledge about particular techniques . The course contains 5 modules, where each module contains 47 recorded lectures (635 min / lecture). Each module contains in total 12.5 h of recorded materials, and all lectures are in english . Each of the 5 modules is followed by a quiz in the format of multiple choice questions . After completing the course, the student should be able to: discuss the nomenclature and subgroups of evs, describe the release and uptake mechanisms of evs, describe the rna, protein and lipid content of evs, explain the considerations that are important during the collection and isolation of evs from different body fluids, describe the basic concepts about the most common isolation and characterisation techniques and how these techniques are used in the ev field andstate the benefits and limitations of the most common isolation and characterisation techniques for evs . Discuss the nomenclature and subgroups of evs, describe the release and uptake mechanisms of evs, describe the rna, protein and lipid content of evs, explain the considerations that are important during the collection and isolation of evs from different body fluids, describe the basic concepts about the most common isolation and characterisation techniques and how these techniques are used in the ev field and state the benefits and limitations of the most common isolation and characterisation techniques for evs . The initial response to the course has been overall positive with high ratings, and one student commented the course as: this course was really well organized and paced but packed full of a lot of really good information from great sources and leaders in the field . I really didn't know anything about exosomes before i started this course and now i feel like i can even teach the people in my own lab a few tricks . This course was really well organized and paced but packed full of a lot of really good information from great sources and leaders in the field . I really didn't know anything about exosomes before i started this course and now i feel like i can even teach the people in my own lab a few tricks . We are pleased to see this initial feedback to the course and isev will now initiate the work of producing more online courses on other related topics such as the biological functions of evs in health and disease . This course was funded by the international society for extracellular vesicles and supported by grants for pedagogic development from the sahlgrenska academy, university of gothenburg . Ysg is the inventor of patents for using evs as therapeutics, diagnostics and vaccines and is the founder of aeon medix and rosetta exosome and own stock in the company . Jl is the co - owner of patents for using exosomes as therapeutics and is currently an employee of codiak biosciences, inc . In parallel with his position at university of gothenburg.
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In the indian subcontinent oral squamous cell carcinoma (oscc), frequently shows metastasis to cervical lymph nodes and despite the availability of modern treatment modalities, the 5-year survival rate has not changed significantly . Treatment options for head and neck cancers based on multiple factors such as age, sex, tumor site, tnm staging and histopathological grade help as an adjunct to guide therapy, but are not reliable predictors of the outcome . The most accurate clinical predictors for recurrence and metastasis at present are nodal staging (n - staging). Unfortunately, many patients come with advanced stage at presentation and n - stage is not very discriminating in this group of patients . Thus, it becomes essential that other avenues be explored like immunohistochemistry of lesional tissue or plasma levels of the patient to check for prognostic tumor markers . One such marker is osteopontin (opn), a secreted phospho - glycoprotein that binds v3 integrin and some cd44 isoforms . Although, molecular mechanisms are not very clear, opn actions in carcinogenesis and metastasis are linked to the changes in cell migrations, angiogenesis and apoptosis . In tumors of breast, prostate and stomach, the clinical significance of osteopontin expression in t1 and t2 tongue cancers have also been correlated, making it a reliable candidate for assessing prognosis . In oscc cases, opn expressions assessed by using immunohistochemistry (ihc) were found to be progressively higher from normal tissues to epithelial hyperplasia, dysplasia and carcinoma in situ, suggesting the role of opn in oral carcinogenesis . Although, plasma opn level has been considered as a prognostic marker for oscc, it has been also noted that plasma level could also be influenced by various inflammatory and fibrotic processes such as atherosclerosis, bone remodelling, angiogenesis, wound healing and tissue injuries . Literature also confirms that tissue expression of opn is a more reliable way to reflect the fidelity of opn expression by the tumor . Hence, we examined the preoperative plasma level of opn and the opn expression by immunohistochemistry (ihc) in the paraffin - embedded tumor tissues by incisional biopsy of the same patients and evaluated the prognostic significance of opn expression in oscc patients . The control group comprised of ten (n = 10); age and sex matched volunteers from whom, in the form of biopsies, normal mucosae and blood samples for plasma were collected . The study group comprised of 60 patients, of which 84% were males and 16% were females . The age of the patients ranged from 23 - 68 years with average age at 48.67 years, who were clinically and histologically diagnosed for oral squamous cell carcinoma . Clinical evaluation including tnm classification was done and fine needle aspiration cytology (fnac) and histopathological examination confirmed the presence of oral squamous cell carcinoma . The grading was done according to malignancy grading system proposed by anneroth's criteria . Those cases which were histopathologically diagnosed as osccall the patients who were ready to undergo the required surgical procedure . Those cases which were histopathologically diagnosed as oscc all the patients who were ready to undergo the required surgical procedure . Those cases which showed cervical lymph node enlargement due to acute or chronic infection without any clinical evidence of oral malignancythose cases which undergoing treatment for recurrence including chemotherapy and radiotherapy . Those cases which showed cervical lymph node enlargement due to acute or chronic infection without any clinical evidence of oral malignancy those cases which undergoing treatment for recurrence including chemotherapy and radiotherapy . Thick tissue sections of 4 m were used for immunohistochemistry procedure . For negative control, positive opn immunostaining was defined as detectable immunoreactivity in the perinuclear and/or other cytoplasmic regions in atleast 10% of the cancer cells . The enzyme linked immunosorbent assay (elisa) technique was used to quantify opn in k - edta plasma from oscc patients and in respective controls . The plasma levels of opn were determined with the human opn elisa kit according to the manufacturer's instructions . After short vortex and visual check, samples were centrifuged at 5000 rpm and 4c . Assay buffer provided in elisa kits was used to dilute plasma samples to a desired proportion . Each sample was tested in duplicate and the results were quantified using a standard curve . The control group comprised of ten (n = 10); age and sex matched volunteers from whom, in the form of biopsies, normal mucosae and blood samples for plasma were collected . The control group comprised of ten (n = 10); age and sex matched volunteers from whom, in the form of biopsies, normal mucosae and blood samples for plasma were collected . The study group comprised of 60 patients, of which 84% were males and 16% were females . The age of the patients ranged from 23 - 68 years with average age at 48.67 years, who were clinically and histologically diagnosed for oral squamous cell carcinoma . Clinical evaluation including tnm classification was done and fine needle aspiration cytology (fnac) and histopathological examination confirmed the presence of oral squamous cell carcinoma . Those cases which were histopathologically diagnosed as osccall the patients who were ready to undergo the required surgical procedure . Those cases which were histopathologically diagnosed as oscc all the patients who were ready to undergo the required surgical procedure . Those cases which showed cervical lymph node enlargement due to acute or chronic infection without any clinical evidence of oral malignancythose cases which undergoing treatment for recurrence including chemotherapy and radiotherapy . Those cases which showed cervical lymph node enlargement due to acute or chronic infection without any clinical evidence of oral malignancy those cases which undergoing treatment for recurrence including chemotherapy and radiotherapy . Thick tissue sections of 4 m were used for immunohistochemistry procedure . For negative control, positive opn immunostaining was defined as detectable immunoreactivity in the perinuclear and/or other cytoplasmic regions in atleast 10% of the cancer cells . The enzyme linked immunosorbent assay (elisa) technique was used to quantify opn in k - edta plasma from oscc patients and in respective controls . The plasma levels of opn were determined with the human opn elisa kit according to the manufacturer's instructions . After short vortex and visual check, samples were centrifuged at 5000 rpm and 4c . Assay buffer provided in elisa kits was used to dilute plasma samples to a desired proportion . Each sample was tested in duplicate and the results were quantified using a standard curve . The study consisted of a cohort of 60 oscc patients and 10 normal patients with average age at 48.67 years . The tumor site, divided the subjects into four categories; buccal mucosa, gingiva, lips and tongue . The tumor sites at buccal mucosa and gingiva were contributing 90% of the total cases . The following chart displays the distribution of cases in oscc cohort according to clinical sites [figure 1]. The oscc cohort was then divided into three categories according to histopathological grading (well - differentiated, moderately differentiated, poorly differentiated) [figure 2] and clinical staging was also done (tnm staging) [table 1]. The habit parameter listed oscc cohorts for tobacco chewing (t), smoking (s) and alcohol (a). The t category (tobacco chewing in various forms including gutkha, mawa, etc) the clinical site distribution of the cases of oscc, where buccal mucosa and gingiva contributed to 90% of the total cases the oscc cohort was divided according to histo - pathological grading distribution of cases according to clinical staging using tnm classification distribution of cases according to habit (tobacco, smoke, alcohol) the comparative evaluation of distribution of oscc and normal cohorts was done from the reading of plasma opn samples . The oscc cohort was distributed with mean opn concentration at 0.11 and standard deviation of 0.049 . The control group was found to be distributed with mean opn expression at 0.10 and standard deviation of 0.035 . The mean plasma opn concentration in oscc cohort was more in comparison to the normal cohort [graph 1]. The distribution of the plasma opn levels in oscc cohorts was then compared with respect to histopathological grades viz . The comparison clearly showed that the plasma opn levels in poorly differentiated grade were higher compared to those from moderately and well differentiated grades [graph 2]. The oscc cohort afterwards, was divided into two categories according to their age viz . The mean opn expression in 50 years was 0.10 compared to 0.13 in the 51 + year category . The mann - whitney u test showed a significant difference in opn expression between the two groups with p value of 0.048 [figure 3]. The box - plot showing comparative distribution of oscc and normal cohorts the distribution of the plasma opn levels in oscc cohorts were compared with respect to histo - pathological grades viz . Well, moderate and poorly differentiated oscc cohort was divided into two categories according to their age viz . <= 50 years and 51 years & above and correlated with opn expression for opn expression quantification using immunohistochemistry (ihc) on tissue samples, the number of cytoplasmic positive squamous neoplastic cells were counted under light microscopy using grizzle et al . Cases were assessed along with its histopathological grading and accordingly its positivity was enumerated relating to intensity [table 3]. When evaluated opn expression had higher intensity observed in oscc (95% + ve) cases than in normal epithelium (ne - 60% + ve) cases [figure 5]. The average total score for opn expression in the oscc cases was 1.65 1.05, whereas in ne it was 0.72 0.51 [graph 3]. Immunostaining for osteopontin expression in oral squamous cell carcinoma with positive osteopontin immunoreactivity seen in the cytoplasm of the neoplastic squamous epithelial cells (ihc, 200) percentage positivity of various grades of intensity distribution observed in oscc and normal cases (a) photomicrograph showing invasion and well differentiated squamous cell carcinoma (h&e, 100) (b) photomicrograph of same section showing positive immunostaining for opn (h&e, 100) (c) photomicrograph showing nuclear and cellular pleomorphism with mitotic figures in the invading islands (h&e, 40) (d) photomicrograph of same section exhibiting characteristic cytoplasmic opn immunostaining in neoplastic cells (ihc, 400) comparision of opn intensity between oscc and control cohorts in the present study, an effort was made to evaluate the efficacy of plasma opn and tissue opn expressions, as a prognostic marker in 60 cases of oscc with different histological grading and 10 cases of ne . Semi - quantitative evaluation of immunopositive reaction of opn and scoring for each case revealed that opn expression was significantly higher in carcinoma cells than in normal . As there was significant difference between oscc and normal cohorts (p <0.05 sig), the value suggests that opn expression increases with malignant transformation . These results were consistent with the study by muramatsu et al ., showing that proliferation and invasion were reduced by inhibition of opn in bsc - of cells . According to our study that reported by us earlier, the authors have concluded and highlighted opn's role as a biomarker for malignancy in the form of invasion using ihc . Plasma levels of opn in patients with head and neck cancer have been found to be elevated and correlate with progression . In another study, devoll et al . Reported that opn was not detected in normal oral mucosa but was detectable in a significant percentage of the tissues with dysplasia, carcinoma in situ and squamous cell carcinomas of oral epithelium by ihc study . In our study, there was a distinct increase of plasma opn in oscc patients clearly indicating its association with its aggressive behavior, which has already been established by chien et al . Moreover, opn levels in patients with oscc were higher than in controls . In the present study, levels of opn were significantly lower in oscc patients with tumor stage t1 compared to those with stage t2 or stages t3 - 4 with the medians 31, 82 and 116 g / l respectively, which simulated with the clinical staging study by chien et al . Oscc patients with tumor grades i - ii had lower concentrations of opn in plasma compared to those with grade iii with the medians 116 g / l . This close correlation with clinico - pathological data clearly indicates that plasma opn is associated with tumor progression in oscc patients . Various malignancies, such as esophageal squamous cell carcinoma, hepatocellular carcinoma, metastatic breast cancer and gastric cancer, have already established significant correlations between plasma opn levels and tumor aggressiveness . This over expression of plasma opn in our study is corroborative of the malignant phenotype, by increasing cell transformation, migration and invasion and is associated with oncogenesis and dissemination of various cancers . In our ihc study, opn expression also significantly correlated with more aggressive tumor behavior in the cohort of oscc patients . In conclusion, high expression level of opn in either the tumor or the plasma of the oscc patients is associated with tumor progression.
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Antiphospholipid syndrome (aps) is an autoimmune disease characterized by the presence of antiphospholipid antibodies, venous and/or arterial thrombosis, and repetitive fetal loss [1, 2]. The laboratory criteria to diagnose aps are high serum levels of lupus anticoagulant as well as anticardiolipin and -2 glycoprotein - i antibodies . Aps can occur with other systemic diseases such as systemic lupus erythematosus or as a single entity . Deep vein thrombosis represents the most frequent aps complication, as it occurs in 2955% of patients; however, acute or indolent chronic ischemic presentations can involve any organ including the lung, skin, brain, liver, kidneys, adrenal glands, heart, and eyes . Ocular involvement can occur in 888% of patients [5, 6, 7] and it can be the first sign at presentation . It is therefore important to make a quick diagnosis in order to consider anticoagulant treatment to prevent further thrombosis, as the risk of this complication is up to 29% in untreated aps patients . Visual symptoms at presentation vary from a reduction of visual acuity and amaurosis fugax to transient scotoma and visual field defect, all of which can be unilateral or bilateral . It is important to underline that, when amaurosis fugax involves both eyes simultaneously, it is often a manifestation of central nervous system ischemia . Ocular discomfort, conjunctival hyperemia, and pain have often been reported in the literature as common symptoms of aps patients . A 38-year - old female patient presented with reduced vision in the right eye, which had occurred the same day . The patient had a negative ocular and medical history and no previous miscarriage as well as no relevant family history for ophthalmic pathologies . Visual acuity was finger count in the right eye (1-meter distance) and 20/20 in the left eye . Fundoscopic examination revealed a subhyaloid hemorrhage, engorged and tortuous retinal veins, intraretinal hemorrhages, and cotton wool spots (fig . 1). Fundus fluorescein angiography demonstrated a delayed inferior hemiretinal venous filling (fig . 3). As choroidal ischemia has been previously reported with aps, indocyanine green angiography was performed, which did not show any significant modification of choroidal perfusion . Prothrombotic conditions secondary to other factors such as sepsis, homocystinemia, and genetic defects of coagulation factors (thrombin mutations, factor v leiden, antithrombin deficiency, etc .) Were ruled out . Laboratory investigations showed that the erythrocyte sedimentation rate, igg anticardiolipin, and -2 glycoprotein - i antibody were above normal limits . Thromboplastin inhibition test was strongly positive for lupus anticoagulant, while antinuclear antibody and antinative dna antibodies were negative . Serum total complement (c3, c4), proteins s and c, and antithrombin iii were within normal limits . Other studies were normal or negative, including complete blood count, platelet count, renal, liver, and thyroid functions, hemoglobin a1c, ferritin, transferrin, and ldl cholesterol . The patient met the criteria for aps; however, according to the american college of rheumatology classification criteria, a concomitant diagnosis of systemic lupus erythematosus could not be established . She started treatment with hydroxychloroquine 200 mg / daily and ticlopidine 250 mg / daily . Considering the central location of the hemorrhage, we deferred any invasive treatment and opted for watchful waiting . The subhyaloid hemorrhage resolved spontaneously and the patient recovered a visual acuity of 20/20 in her right eye 3 months after the initial episode (fig . This is the first reported case of simultaneous subhyaloid hemorrhage and branch retinal vein subocclusion as presentations of aps . A prompt diagnosis of this condition is fundamental to consider a systemic treatment to avoid any further thrombosis . This is often difficult, as ocular manifestations of aps can be variable . Assessing the patient's thrombotic risk by investigating the presence of high blood pressure and hypercholesterolemia, the use of tobacco and oral anticontraceptive agents as well as the history of previous of thrombosis, fetal loss, and acute ischemic events is mandatory in the process of deciding whether to start systemic treatment.
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The prevalence of kidney stones in the united states has risen steadily in recent years and correlates with population dietary changes . Relative dietary intake of fluid, sodium, calcium, oxalate, citrate, and animal - based proteins has a marked influence on stone risk . Accordingly, an efficient assessment of a patient's nutritional intake is important for the urologist and may enable an informed assessment and guided recommendations . The traditional office based interview may not adequately assess the patient's nutritional intake, due to time constraints and the detailed nature of a nutritional history . Furthermore, biases may exist both in how a patient wishes to represent themselves, and in how the practitioner presents a question . We sought to determine if a physician's impression of a patient's dietary intake was dependent on the medium through which they obtained the nutritional data . A typical office interview was compared to information obtained via a computerized clinical decision support system (cdss), which presented a food frequency questionnaire (ffq). Furthermore, the urologists made recommendations for dietary intervention based on the information ascertained via each method . Each patient completed: (1) an office - based interview with one of two fellowship - trained endourologists, and (2) a ffq administered via a cdss, presented on an ipad . Each urologist made two separate assessments via a likert - scale - based survey of the significance of a patient's nutritional (nutritional impact score) and hydration factors (hydration impact score) as they pertained to stone disease (fig . Each assessment was followed by specific recommendations, such as reducing sodium intake, reducing animal protein intake, and increasing fluid intake . The first assessment (pre - ffq) was made after the office - based interview with the patient but without reviewing the results of the ffq . The second assessment (post - ffq) was made after reviewing the patient's ffq results . If the urologist modified the nutritional impact score, hydration impact score, or treatment recommendations from the first assessment to the second, we assumed that the ffq results caused the urologist to believe that nutrition had a greater or lesser impact on the patient's stone disease than previously assessed by the office - based interview . The ffq used in this study is an 88-question nutritional survey that is similar to prior ffqs that have been validated by previous analyses for accurate assessment of a patient's dietary habits . Using the ffq, our patients recorded nutritional intake of those foods providing substantial quantities of the following: oxalate, animal - based protein, sodium, and calcium . Intake of fruits and vegetables, fluids, and nutritional supplements was also assessed (fig . Questionnaire results were entered in the ffq's algorithm, which generated numerical scores corresponding to patient's relative intake in each of the following domains: oxalate, calcium, sodium, purine, and fluids . For each patient, we compared pre - ffq nutritional impact score, hydration impact score, and treatment recommendations to respective post - ffq data points . Our results were coded into the following binary variables, detailing the presence or absence of: increase in diet score, increase in hydration impact score, any change in treatment recommendations, addition of at least one treatment recommendation, and subtraction of at least one treatment recommendation . A multivariate logistic regression model was fit to determine the impact of the numerical results of each domain in the ffq (oxalate, calcium, sodium, purine, and fluids) and the aforementioned binary variables . Each patient completed: (1) an office - based interview with one of two fellowship - trained endourologists, and (2) a ffq administered via a cdss, presented on an ipad . Each urologist made two separate assessments via a likert - scale - based survey of the significance of a patient's nutritional (nutritional impact score) and hydration factors (hydration impact score) as they pertained to stone disease (fig . Each assessment was followed by specific recommendations, such as reducing sodium intake, reducing animal protein intake, and increasing fluid intake . The first assessment (pre - ffq) was made after the office - based interview with the patient but without reviewing the results of the ffq . The second assessment (post - ffq) was made after reviewing the patient's ffq results . If the urologist modified the nutritional impact score, hydration impact score, or treatment recommendations from the first assessment to the second, we assumed that the ffq results caused the urologist to believe that nutrition had a greater or lesser impact on the patient's stone disease than previously assessed by the office - based interview . The ffq used in this study is an 88-question nutritional survey that is similar to prior ffqs that have been validated by previous analyses for accurate assessment of a patient's dietary habits . Using the ffq, our patients recorded nutritional intake of those foods providing substantial quantities of the following: oxalate, animal - based protein, sodium, and calcium . Intake of fruits and vegetables, fluids, and nutritional supplements was also assessed (fig . Questionnaire results were entered in the ffq's algorithm, which generated numerical scores corresponding to patient's relative intake in each of the following domains: oxalate, calcium, sodium, purine, and fluids . For each patient, we compared pre - ffq nutritional impact score, hydration impact score, and treatment recommendations to respective post - ffq data points . Our results were coded into the following binary variables, detailing the presence or absence of: increase in diet score, increase in hydration impact score, any change in treatment recommendations, addition of at least one treatment recommendation, and subtraction of at least one treatment recommendation . A multivariate logistic regression model was fit to determine the impact of the numerical results of each domain in the ffq (oxalate, calcium, sodium, purine, and fluids) and the aforementioned binary variables . Between the 2 urologists in the study, there was no significant difference in the average nutritional impact scores or average the hydration impact scores assigned . From pre - ffq to post - ffq, the urologist was more likely to increase the nutritional impact score if the patient had higher ffq scores for sodium (odds ratio [or], 1.02; p=0.02) or fluids (or, 1.03, p=0.04). In other words, the physician interpreted a greater contribution of diet to the patient's stone disease when the ffq indicated higher sodium or fluid intake (table 2). There was no difference in hydration score rendered by the physician for patients who consumed increased oxalate, calcium, sodium, purine, or fluids (table 3). A higher ffq score for oxalate was positively associated with the addition of at least one treatment recommendation (or, 1.07; p=0.02) (table 4). Stone disease is among the most common and costly urological conditions, with a lifetime prevalence of approximately 9%, and 5-year recurrence rates approaching 50% . In the united states alone, the economic burden of stone disease has been estimated to be over $2 billion annually . Previous studies have underscored the impact of diet on stone formation and the role of dietary interventions in prevention of stone disease . Our study revealed that using a ffq to obtain the diet history impacted how urologists perceived the effects of diet and hydration on stone disease, as well as how urologists treated the stone disease . Therefore, it is important for the urologist to effectively and expeditiously obtain a thorough diet history . Certainly, the modern urologist experiences logistical and time constraints in his or her busy clinical practice . Additionally, some patients may alter their responses to dietary questions in an attempt to represent their habits more favorably . Furthermore, even skilled endourologists and stone experts may not use the most effective questions to obtain the information necessary . Finally, a comprehensive stone clinic, complete with a dietician is favorable, but not feasible in every practice setting . Previous authors have found the ffq to be an effective tool for obtaining an accurate and objective diet history . As for the method of delivering the ffq, we believe that the computerized cdss provides physicians with a relatively bias - free mode of data collection as well as standardized evaluations of the data delivered in a timely fashion . In other medical applications, the cdss has been shown to improve practitioner performance and patient care, for instance, by reducing medication errors . In the present study, we found that the use of the ffq as a cdss can significantly alter the how the endourologist assesses the significance of dietary factors on a patient's stone disease . Moreover, the recommended dietary interventions can change based on the results of the ffq . In our urolithiasis clinic, nearly all of the patients (75 of 76) were willing to use the ffq . These findings suggest that the addition of the ffq to the 24-hour urine collection and other components of the comprehensive stone evaluation may alter the assessment and plan for these patients . The major limitation of our study is that we did not determine if use of the ffq leads to improved patient outcomes, such as decreased recurrence rates or increased patient satisfaction . Future studies may be developed to link the use of the ffq to improved outcomes or patient encounters . While our study may not yet translate into improved patient outcomes, certainly others have shown that the use of cdss can improve efficiency . It is reasonable to hypothesize that improving efficiency may be a key component in improving patient outcomes . Other studies have also shown that ffqs can play an integral role in assessing urolithiasis patients, especially in a multidisciplinary setting, where a patient's diet may be a key focus in stone prevention . While our ffq was reasonably detailed, others have been able to streamline a more lengthy diet questionnaire into a more compact rapid food screener . In regards to assessing renal acid load in stone formers, his goal was to make this rapid assessment equivalent to a more lengthy diet questionnaire . In a follow - up study, patients were asked to complete both of these questionnaires, and their lake score assessment appeared as good as a lengthier questionnaire in determining renal acid load . While this particular study looked at a different aspect of stone disease, it appears that endourologists are attempting to gain detailed dietary information from their patients while continuing to improve efficiency and streamline their stone clinics . Certainly these approaches may prove worthwhile as we continue to develop this tool in our urolithiasis clinic . We hope that our efforts may ultimately improve the efficiency and quality of care of our endourology patients . Given the wide body of evidence supporting the use of dietary interventions in preventing stone recurrence, it is reasonable to hypothesize that recommendations made with potentially more complete and accurate data, obtained using the ffq, would confer improved outcomes . Randomized - controlled trials are necessary to evaluate the impact of utilizing the ffq as a cdss on stone patients to patient outcomes, such as recurrence rates, quality of life, and satisfaction with the clinical experience . A self - reported, electronic version of the ffq may be helpful in the efficient assessment and counseling of patients with stone disease . With prior comprehensive knowledge of a patient's nutritional intake, the urologist may be afforded more time with which to counsel patients regarding intervention and nutritional intake.
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Delirium is an acute complex neuropsychiatric condition, which is highly prevalent in inpatient treatment setting and is considered to lead to many adverse outcomes in medically ill patients . Although many studies have evaluated the phenomenology of delirium, very few studies have evaluated the relationship of cognitive and noncognitive symptoms . Existing data suggest that noncognitive features dominate the clinical picture during the early stages of delirium while cognitive impairments peak after 1 week and dominate the clinical picture thereafter . In another study, the authors evaluated patients with delirium on mini - mental status examination (mmse), delirium rating scale (drs), and memorial delirium assessment scale (mdas) and reported high correlations between individual mdas items and the mmse total score when compared with the correlation between individual drs items and the mmse score . Noncognitive items (e.g., perceptual disturbances, sleep - wake cycle disturbances) of both scales and certain specific drs items (i.e., lability of mood, physical disorder) showed lower correlations with the mmse . A previous study from our center evaluated the patients of delirium rating scale - revised 1998 (drs - r-98) and cognitive test for delirium (ctd) and showed that patients experiencing higher attention deficits had higher dysfunction on all other domains of cognition on ctd . In terms of the relationship between cognitive and noncognitive functions, it was seen that cognitive functions as assessed on ctd correlated positively with and total drs - r-98 score, drs - r-98 severity score, and drs - r-98 severity score without the attention item score . However, there were few correlations between various domains scores and ctd total scores with cognitive domain score of drs - r-98 (items 913) and noncognitive symptom total score of drs - r-98 (items 18). The commonly used instrument for assessment of cognitive functions includes mini - mental state examination (mmse) and ctd and the cognitive subscale of delirium rating scale - revised 1998 version . However, the use of mmse is criticized for being only useful in cooperative patients, its practice effect and influence of level of education . On the other hand, ctd is considered to be more comprehensive which can be used in nonverbal intensive care unit patients . However, its use requires slightly higher level of cooperation of the patient which makes it use difficult in routine clinical situations . In view of these, there is always an effort to evaluate the cognitive functions of patients with delirium using more standardized instruments . Accordingly, there is a need to understand the suitability of instrument which can be used to assess cognitive functions among patients with delirium . Studies done in india involving patients of dementia also suggest that mmse is not a good instrument to evaluate cognitive functions among indians, especially those who are illiterate . Considering these limitations, researchers have developed hindi mental status examination (hmse), which has been shown to be possibly more useful for illiterate subjects . Accordingly, the aims of this study were: (1) to evaluate the cognitive functions of patients with delirium using hmse; (2) to evaluate the correlation of cognitive functions assessed by hmse with noncognitive symptoms as assessed using drs - r-98; (3) to study the association of cognitive functions assessed using hmse and drs - r-98 . Participants were recruited after obtaining written informed consent from the family caregivers, staying with the patients during the inpatient stay . All the patients referred to psychiatry consultation - liaison (cl) psychiatric services during the study period of august 2014 to december 2014 and diagnosed with delirium were eligible for this study . For inclusion into the study, the patients were required to be aged 15 or more and not on any antipsychotic medications before assessment . Patients with loss of vision, hearing impairment, aphasia, axis - i psychiatric disorders were excluded from this study . The presence of preexisting cognitive deficits was ruled out on the basis of short informant questionnaire on cognitive decline in the elderly (retrospective iqcode). Those admitted to the intensive care units and on mechanical ventilation were excluded from this study . Each severity item is rated on a 4 point scale (03), and the mean severity score ranges from 0 to 39 . It has been shown to have high inter - rater reliability, sensitivity, and specificity . It is based on mmse and assesses the same domains of cognitive functions as mmse, however, the items are designed by taking the literacy level and cultural issues into account . It has been used by many researchers for screening patients for dementia and also has been used in patients with delirium but the authors did not present the detail scores . It is a 16-item instrument which on the basis of the input provided by a key relative determines the cognitive status of the patient for the specified period (6 months for this study). Each item is rated on a 5-point scale with score 3 indicating no change, and scores higher than 3 indicating decline in cognitive functioning and lower scores indicates improvement in cognitive functions . To obtain the final score, score obtained for all items is added and divided by 16 . A mean score> 3.313.38 is considered to be an indicator of cognitive decline . Family carers of the patients diagnosed with delirium by the cl psychiatry team were approached and explained about the purpose of the study . Initially, the consenting caregivers were interviewed to confirm in the patient the diagnosis of delirium as per diagnostic and statistical manual of mental disorders-4 edition text revision criteria and to rule out other psychiatric disorders . Then the patients with confirmed diagnosis of delirium were evaluated on short iqcode to rule out preexisting cognitive deficits . All the assessment was done over a period of 1 h with breaks in between . Chicago, spss inc . ). For descriptive purposes mean, standard deviation (sd), frequency and percentages were calculated . Each severity item is rated on a 4 point scale (03), and the mean severity score ranges from 0 to 39 . It has been shown to have high inter - rater reliability, sensitivity, and specificity . It is based on mmse and assesses the same domains of cognitive functions as mmse, however, the items are designed by taking the literacy level and cultural issues into account . It has been used by many researchers for screening patients for dementia and also has been used in patients with delirium but the authors did not present the detail scores . It is a 16-item instrument which on the basis of the input provided by a key relative determines the cognitive status of the patient for the specified period (6 months for this study). Each item is rated on a 5-point scale with score 3 indicating no change, and scores higher than 3 indicating decline in cognitive functioning and lower scores indicates improvement in cognitive functions . To obtain the final score, score obtained for all items is added and divided by 16 . A mean score> 3.313.38 is considered to be an indicator of cognitive decline . Family carers of the patients diagnosed with delirium by the cl psychiatry team were approached and explained about the purpose of the study . Initially, the consenting caregivers were interviewed to confirm in the patient the diagnosis of delirium as per diagnostic and statistical manual of mental disorders-4 edition text revision criteria and to rule out other psychiatric disorders . Then the patients with confirmed diagnosis of delirium were evaluated on short iqcode to rule out preexisting cognitive deficits . All the assessment was done over a period of 1 h with breaks in between . Each severity item is rated on a 4 point scale (03), and the mean severity score ranges from 0 to 39 . It has been shown to have high inter - rater reliability, sensitivity, and specificity . It is based on mmse and assesses the same domains of cognitive functions as mmse, however, the items are designed by taking the literacy level and cultural issues into account . It has been used by many researchers for screening patients for dementia and also has been used in patients with delirium but the authors did not present the detail scores . It is a 16-item instrument which on the basis of the input provided by a key relative determines the cognitive status of the patient for the specified period (6 months for this study). Each item is rated on a 5-point scale with score 3 indicating no change, and scores higher than 3 indicating decline in cognitive functioning and lower scores indicates improvement in cognitive functions . To obtain the final score, score obtained for all items is added and divided by 16 . A mean score> 3.313.38 is considered to be an indicator of cognitive decline . Family carers of the patients diagnosed with delirium by the cl psychiatry team were approached and explained about the purpose of the study . Initially, the consenting caregivers were interviewed to confirm in the patient the diagnosis of delirium as per diagnostic and statistical manual of mental disorders-4 edition text revision criteria and to rule out other psychiatric disorders . Then the patients with confirmed diagnosis of delirium were evaluated on short iqcode to rule out preexisting cognitive deficits . All the assessment was done over a period of 1 h with breaks in between . Chicago, spss inc . ). For descriptive purposes mean, standard deviation (sd), frequency and percentages were calculated . The mean age of the study participants was 47.10 (sd - 16.5) years with a range of 1575 years . About one - fifth (n = 18; 23.68%) of the study sample was aged 60 years or more . The majority of the participants were male (n = 55; 72.4%) and the mean duration of delirium at the time of assessment was 2.23 (sd - 3.1) days . The symptom profile as per drs - r-98 all the patients had sleep - wake cycle disturbances, acute onset of illness, fluctuations of symptoms, and presence of a physical disorder . The mean drs - r-98 score 33.9 (sd - 7.2) and the mean drs - r-98 severity score was 25.9 (sd - 7.2). The mean score of noncognitive domain was more than mean score of cognitive domain . Frequency of delirium symptoms on drs - r-98 as per levels of severity in the included group the mean score on hmse was 19.3 (7.98). Is considered to be the core cognitive symptoms of delirium, based on the attention scores obtained on hmse, the study sample was divided into three groups, i.e., those who scores 01, 23, and 45 and the three groups were compared for the drs - r-98 score and scores in the other domains of hmse [table 2]. Comparison of severity of delirium symptoms on drs - r-98 as per levels of severity of attention deficits on hmse as shown in table 3, there were significant correlations of all the domains of hmse with drs - r-98 total score, drs - r-98 severity score, drs - r-98 cognitive subscale score, drs - r-98 noncognitive domain subscale score and drs - r-98 severity score without attention score . Association of scores on drs - r98 and hmse when the association of each item of drs - r-98 and hmse was evaluated, except for the items of delusions, lability of affect and motor retardation, there were significant negative association between all the items of drs - r-98 and hmse, indicating that higher severity of cognitive symptoms as assessed on hmse is associated with higher severity of all the cognitive symptoms and most of the noncognitive symptoms as assessed by drs - r-98 . As shown in table 3, there were significant correlations of all the domains of hmse with drs - r-98 total score, drs - r-98 severity score, drs - r-98 cognitive subscale score, drs - r-98 noncognitive domain subscale score and drs - r-98 severity score without attention score . Association of scores on drs - r98 and hmse when the association of each item of drs - r-98 and hmse was evaluated, except for the items of delusions, lability of affect and motor retardation, there were significant negative association between all the items of drs - r-98 and hmse, indicating that higher severity of cognitive symptoms as assessed on hmse is associated with higher severity of all the cognitive symptoms and most of the noncognitive symptoms as assessed by drs - r-98 . The sample included in the present study is similar to the previous studies from cl psychiatry set - up from india, in terms of mean age, percentage of elderly, gender distribution, mean duration of education, duration of delirium, and mean number of associated etiologies suggesting that the study sample was representative . The drs - r-98 symptom profile of the study sample is also similar to previous studies from this center, both in terms of frequency and severity of symptoms and literature from other parts of the world . In terms of associations of various symptoms domains, considering the fact that attention is the most important cognitive dysfunction in patients with delirium, we evaluated the correlation between attention and other cognitive functions as assessed by hmse . It was seen that there was positive association between attention and all other cognitive domains as assessed by hmse, suggesting that higher attention deficits are associated with higher dysfunction on all other domains . Previous studies have also evaluated the similar associations, however, using ctd and have reported similar positive association . In terms of the influence of attention as assessed by using hmse on other symptoms of delirium as assessed by drs - r-98, findings of the present study suggest that attention has a significant influence on most of the other symptoms of delirium too, with higher level of attention associated with higher level of dysfunctions in other symptom domains . Previous studies which have evaluated the influence of attention as assessed by ctd are not conclusive . One study from the united kingdom, reported significant influence of attention deficits as assessed by ctd on sleep - wake cycle disturbances, language, through process disturbance, orientation, short - term memory, long - term memory, visuospatial abnormalities, and total drs - r-98 scores . However, the previous study done at our center using the similar scales used in the study from the united kingdom did not find such associations except for association of attention and motor retardation . Accordingly, it can be said that as suggested in previous study from the united kingdom, attention deficits in patients of delirium influences other symptoms domain too . More clear influence of attention deficit noted in the present study could be due to use of a validated and culturally sensitive instrument to assess cognitive functions . This also possibly suggests that in indian set - up hmse may be a better instrument to assess cognitive functions than ctd . When the association of hmse and each item of drs - r-98, drs - r-98 total score, drs - r-98 severity score, drs - r-98 cognitive and noncognitive domains were evaluated, there was significant association of all the domains of cognitive functions assessed by hmse with most of the drs - r-98 items and all the total scores . The findings of an association of total hmse score and various total scores of drs - r-98 suggests that increasing severity of delirium is associated with higher level of cognitive dysfunction . However, the previous study did not find such clear associations for each item of drs - r-98 as noted in this study . In contrast to the previous study from india which did not find an association between cognitive domains of ctd and drs - r-98, the present study showed that there was a significant association between the hmse domains and various cognitive functions as assessed using drs - r-98 . This finding also possibly suggests that hmse may be better instrument for evaluation of cognitive functions in the indian set - up . The present study has certain limitations, which include cross - sectional evaluation of the patients, small sample size and inclusion of etiologically heterogeneous group . As the study did not include patients admitted to intensive care units, the findings cannot be generalized to this group of patients . Assessment of cognitive functions in the present study was limited to the use of hmse and drs - r-98 . Accordingly, it cannot be conclusively suggested that hmse is better than other instruments for assessment of cognitive functions in patients of delirium . Future studies also must compare the use of various instruments such as mmse, hmse, ctd, and montreal cognitive assessment for assessment of cognitive functions among patients with delirium . The present study suggests that attention deficits in patients with delirium influence the severity of cognitive and noncognitive symptoms of delirium . Further, the present study suggests an increase in the severity of cognitive symptoms in other domains is also associated with increase in severity of noncognitive symptoms of delirium . The present study also provides some credence to the fact that hmse may be a better instrument to assess cognitive functions of patients with delirium in the indian setting.
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Although the arterial grafts have superior long - term patency rates, especially for left internal mammary artery to left anterior descending artery, saphenous vein grafts continue to be the backbone of daily coronary revascularization practice . The sequential vein bypass grafting is a frequently used technique in coronary surgical revascularization whereby more than one distal anastomosis is constructed per segment of conduit used . The major advantages of sequential vein bypass grafting includes saving graft material, reduction of the number of proximal anastomoses and more importantly, higher graft flow and thereby, increased graft patency rates . Furthermore, by allowing anastomoses to smaller coronary arteries in patients with diffuse coronary disease, sequential vein bypass grafting is thought to achieve a more complete revascularization, which theoretically should translate into better clinical outcomes . In the present era of coronary artery surgery, an increasing volume of patients with diffuse coronary disease and patients requiring re - do coronary artery bypass grafting (cabg) with limited available graft material are being referred to surgical revascularization since the number of patients who underwent cabg over the past four decades is relatively huge and patients with focal coronary artery lesions or even simple multi - vessel coronary diseases are being successfully treated with percutaneous coronary intervention (pci). Under this situation, however, there are inconsistent and limited evidences with regard to clinical outcomes of sequential vein bypass grafting . Moreover, most of those studies were conducted two decades ago in patients undergoing on - pump cabg and no longer reflect contemporary coronary surgical management . Less is known about the impact of sequential vein bypass grafting on clinical outcomes in off - pump cabg . So the objective of this study is to provide a present - day assessment of the effects of sequential vein bypass grafting on in - hospital and mid - term clinical outcomes following off - pump cabg . A cohort of 920 consecutive patients, who underwent isolated off - pump cabg from october 2009 to september 2013, operated by one surgical team at the fuwai hospital (beijing, china), was obtained . Sequential venous graft was defined as a venous graft with the number of distal anastomoses exceeding the number of its proximal anastomoses . Of those 920 patients, there were 165 patients with at least one sequential venous graft (sequential venous grafts group) and the remainder with individual venous grafts only (individual venous grafts group). We propensity - matched 127 out of 165 patients receiving at least one sequential venous graft to 127 out of 755 patients with individual venous graft only . This study was approved by the institutional review board of fuwai hospital with patient informed consent waived . Because off - pump cabg is technically more demanding, and expertise in constructing a sequential anastomosis is perhaps among the major determinants of graft patency or clinical outcomes, the present study only includes patients operated by one surgical team to minimize the surgical variability . The heart was exposed through a median sternotomy in all the patients . An octopus stabilizer (medtronic, minneapolis, mn), a humidified carbon dioxide blower (medtronic dlp, grand rapids, mi) and intracoronary shunts (cardiothoracic system, cupertino, ca, usa) the number of distal anastomoses is generally two on one sequential venous graft in our current policy . The distal anastomosis on a sequential graft was done in end - to - side fashion and middle ones were done in side - to - side fashion . Side - to - side anastomoses were performed in a crossing fashion and in a diamond - shape, and end - to - side ones were done parallel to the native coronary vessel axis . All distal anastomoses were done using double - armed 70 polypropylene sutures with a continuous suturing technique . Proximal anastomoses were stitched to the ascending aorta with continuous double armed 60 polypropylene sutures during partial - clamping of the aorta . When hemodynamics were stable after completion of all the anastomoses, the graft blood flow and pulsatility index were measured by flow meter to confirm patency of the grafts (veri - q, medistim, oslo, norway). The in - hospital measurement of our study was composite outcome of in - hospital death, myocardial infarction (mi), stroke, the need for intra - aortic balloon pump (iabp) assistance and prolonged ventilation . Major adverse cardiac events (maces: death, mi or repeat revascularization) and angina recurrence during follow - up were considered as mid - term endpoints . In - hospital death perioperative mi was documented by the following criteria: the creatine kinase mb must be 5 times the upper limit of normal or development of new q waves in two or more contiguous electrocardiograph leads . We excluded confused states, transient events, and intellectual impairment to avoid any subjective bias . Death during follow - up was defined as all - cause mortality after patient's discharge . Mi during follow - up was defined a new q wave in two or more contiguous leads on electrocardiography, or significant increase of cardiac enzyme levels (great than the upper limit of the normal) combined with electrocardiographic or clinical or angiographic evidence of mi . Recurrence of angina was defined as occurrence of chest pain or distress due to myocardial ischemia after discharge . The postoperative results were assessed in all patients at discharge and during follow - up . Patients diagnosed with hyperlipemia resumed statins therapy postoperatively and during follow - up, and beta - blocker and aspirin were generally taken in all the patients except for few patients with respective contraindications . All patients had been followed up at least three months since discharge from hospital with the exception of patients who were lost to follow - up . Follow - up involved review of outpatient and (or) inpatient medical records and structured telephone interviews . The medical records in outpatient clinics of those who reported any adverse events after discharge were reviewed for further confirmation . When any major adverse event was reported by another hospital, patients were requested to mail a copy of all relevant medical information . Follow - up was 95.7% (243/254) complete with a mean follow - up of 22.5 months (with a median follow - up of 23.6 and 21.6 months for maces and angina recurrence respectively). To minimize potential selection bias in the comparisons of outcomes between the two groups, a propensity score matching using multivariate logistic regression model was performed to match patients of sequential venous grafts with patients of individual venous grafts only in a 1-to-1 fashion . Variables selected for inclusion in the propensity score were core patients characteristics, including age, sex, insulin - dependent diabetes, hypertension, body mass index, left ventricular ejection fraction, previous cardiac surgery, numbers of vessels grafted and extent of coronary disease as previously described . Left ventricular end diastolic diameter was also included because of significant difference among the two groups before matching . The receiver operating characteristics curve was used to estimate the area under the curve of the model, predicting the probability of being included in any of the two groups . The c statistic, which is equivalent to the receiver operating characteristic curve, for this model was 0.86 . Using a greedy matching algorithm, patients of sequential venous grafts were matched with patients of individual venous grafts only on an identical propensity score in a higher - digit priority order, namely a five - digit to a four-, three-, two-, or one - digit match . If more than one patient of individual venous grafts only were matched to patient of sequential venous grafts, patient with individual venous grafts only was selected randomly among those patients and eventually, 127 well - matched pairs of patients were obtained . The student's t - test was used to measure the differences for variables with a normal distribution and equal variances . Categorical data are displayed as frequencies and percentages and comparisons were made with chi - square tests (fisher exact tests if appropriate). Kaplan - meier product limit curves for event - free survival were constructed and compared with the log - rank test . The hazard ratio (hr) with 95% confidence interval (ci) all statistical analyses were performed using statistical analysis system (sas) for windows version 9.1 (sas institute, cary, nc). A cohort of 920 consecutive patients, who underwent isolated off - pump cabg from october 2009 to september 2013, operated by one surgical team at the fuwai hospital (beijing, china), was obtained . Sequential venous graft was defined as a venous graft with the number of distal anastomoses exceeding the number of its proximal anastomoses . Of those 920 patients, there were 165 patients with at least one sequential venous graft (sequential venous grafts group) and the remainder with individual venous grafts only (individual venous grafts group). We propensity - matched 127 out of 165 patients receiving at least one sequential venous graft to 127 out of 755 patients with individual venous graft only . This study was approved by the institutional review board of fuwai hospital with patient informed consent waived . Because off - pump cabg is technically more demanding, and expertise in constructing a sequential anastomosis is perhaps among the major determinants of graft patency or clinical outcomes, the present study only includes patients operated by one surgical team to minimize the surgical variability . An octopus stabilizer (medtronic, minneapolis, mn), a humidified carbon dioxide blower (medtronic dlp, grand rapids, mi) and intracoronary shunts (cardiothoracic system, cupertino, ca, usa) were used routinely to facilitate the anastomoses distally or proximally . The number of distal anastomoses is generally two on one sequential venous graft in our current policy . The distal anastomosis on a sequential graft was done in end - to - side fashion and middle ones were done in side - to - side fashion . Side - to - side anastomoses were performed in a crossing fashion and in a diamond - shape, and end - to - side ones were done parallel to the native coronary vessel axis . All distal anastomoses were done using double - armed 70 polypropylene sutures with a continuous suturing technique . Proximal anastomoses were stitched to the ascending aorta with continuous double armed 60 polypropylene sutures during partial - clamping of the aorta . When hemodynamics were stable after completion of all the anastomoses, the graft blood flow and pulsatility index were measured by flow meter to confirm patency of the grafts (veri - q, medistim, oslo, norway). The in - hospital measurement of our study was composite outcome of in - hospital death, myocardial infarction (mi), stroke, the need for intra - aortic balloon pump (iabp) assistance and prolonged ventilation . Major adverse cardiac events (maces: death, mi or repeat revascularization) and angina recurrence during follow - up were considered as mid - term endpoints . In - hospital death perioperative mi was documented by the following criteria: the creatine kinase mb must be 5 times the upper limit of normal or development of new q waves in two or more contiguous electrocardiograph leads . We excluded confused states, transient events, and intellectual impairment to avoid any subjective bias . Death during follow - up was defined as all - cause mortality after patient's discharge . Mi during follow - up was defined a new q wave in two or more contiguous leads on electrocardiography, or significant increase of cardiac enzyme levels (great than the upper limit of the normal) combined with electrocardiographic or clinical or angiographic evidence of mi . Recurrence of angina was defined as occurrence of chest pain or distress due to myocardial ischemia after discharge . The postoperative results were assessed in all patients at discharge and during follow - up . Patients diagnosed with hyperlipemia resumed statins therapy postoperatively and during follow - up, and beta - blocker and aspirin were generally taken in all the patients except for few patients with respective contraindications . All patients had been followed up at least three months since discharge from hospital with the exception of patients who were lost to follow - up . Follow - up involved review of outpatient and (or) inpatient medical records and structured telephone interviews . The medical records in outpatient clinics of those who reported any adverse events after discharge were reviewed for further confirmation . When any major adverse event was reported by another hospital, follow - up was 95.7% (243/254) complete with a mean follow - up of 22.5 months (with a median follow - up of 23.6 and 21.6 months for maces and angina recurrence respectively). To minimize potential selection bias in the comparisons of outcomes between the two groups, a propensity score matching using multivariate logistic regression model was performed to match patients of sequential venous grafts with patients of individual venous grafts only in a 1-to-1 fashion . Variables selected for inclusion in the propensity score were core patients characteristics, including age, sex, insulin - dependent diabetes, hypertension, body mass index, left ventricular ejection fraction, previous cardiac surgery, numbers of vessels grafted and extent of coronary disease as previously described . Left ventricular end diastolic diameter was also included because of significant difference among the two groups before matching . The receiver operating characteristics curve was used to estimate the area under the curve of the model, predicting the probability of being included in any of the two groups . The c statistic, which is equivalent to the receiver operating characteristic curve, for this model was 0.86 . Using a greedy matching algorithm, patients of sequential venous grafts were matched with patients of individual venous grafts only on an identical propensity score in a higher - digit priority order, namely a five - digit to a four-, three-, two-, or one - digit match . If more than one patient of individual venous grafts only were matched to patient of sequential venous grafts, patient with individual venous grafts only was selected randomly among those patients and eventually, 127 well - matched pairs of patients were obtained . The student's t - test was used to measure the differences for variables with a normal distribution and equal variances . Categorical data are displayed as frequencies and percentages and comparisons were made with chi - square tests (fisher exact tests if appropriate). Kaplan - meier product limit curves for event - free survival were constructed and compared with the log - rank test . The hazard ratio (hr) with 95% confidence interval (ci) all statistical analyses were performed using statistical analysis system (sas) for windows version 9.1 (sas institute, cary, nc). The baseline characteristics of the propensity - matched patients with sequential or individual vein bypass grafting respectively are given in table 1 . Both groups were well matched with the only significant difference being that patients receiving sequential grafts had lower rates of preoperative angina (p = 0.03). No significant difference was observed between the two groups in distal anastomoses, graft pulsative index and blood flow per distal anastomoses . Despite of fewer proximal anastomoses (p <0.001), patients with sequential grafts had a higher blood flow per vein graft (p <0.001), especially per sequential vein graft (p <0.001). Baseline characteristics of propensity - matched patients based on sequential venous grafts bmi: body mass index; copd: chronic obstructive pulmonary disease; iabp: intra - aortic ballon pump; lima: left internal mammary artery; lvedd: left ventricular end diastolic diameter; nyha: new york heart association; pci: percutaneous coronary intervention; rwma: regional wall motion abnormity; sd: standard deviation . Procedural characteristics of propensity - matched patients based on sequential venous grafts * values for svg and ivg respectively . Ivg: individual vein graft; svg: sequential vein graft; vg: vein graft . No significant differences was present in in - hospital composite endpoints, with 14 (11.0%) total events in individual venous grafts group versus 18 (14.2%) in sequential venous grafts group (p = 0.45). Also, there were no significant differences between individual venous grafts and sequential venous grafts group with regard to the individual outcome of in - hospital mortality (0.8% vs. 0.0%, p = 1.00), mi (0.8% vs. 0.8%, p = 1.00), stroke (0.8% vs. 0.8%, p = 1.00), iabp assistance (0.0% vs. 0.8%, p = 1.00) and prolonged ventilations (10.2% vs. 13.4%, p = 0.44). In - hospital outcomes of propensity - matched patients based on sequential venous grafts iabp: intra - aortic ballon pump . Composite in - hospital outcome includes in - hospital mortality, myocardial infarction, stroke; iabp requirement and prolonged ventilation . Major adverse cardiac events occurred in six (4.7%) patients (two deaths, two mis and two pcis) in individual venous grafts group and two (1.6%) patients (two mis) in sequential venous grafts group . The kaplan - meier mace - free survival estimates at about four years were 92.5% and 97.3% in the individual and sequential venous grafts group respectively (p = 0.36; figure 1a). The hr for sequential vein bypass grafting was estimated at 0.21 (95% ci, 0.031.85, p = 0.16). The survival rates free of angina recurrence at about 4 years was 80.9% in patients receiving individual venous grafts only versus 85.5% in patients receiving sequential venous grafts (p = 0.48; figure 1b), with hr for sequential vein bypass grafting at 0.70 (95% ci, 0.271.83, p = 0.47). Event - free kaplan - meier estimates for individual venous grafts group and sequential venous grafts group respectivly . Shown are percent survival free from mace (a) and survival free of angina recurrence (b). The baseline characteristics of the propensity - matched patients with sequential or individual vein bypass grafting respectively are given in table 1 . Both groups were well matched with the only significant difference being that patients receiving sequential grafts had lower rates of preoperative angina (p = 0.03). No significant difference was observed between the two groups in distal anastomoses, graft pulsative index and blood flow per distal anastomoses . Despite of fewer proximal anastomoses (p <0.001), patients with sequential grafts had a higher blood flow per vein graft (p <0.001), especially per sequential vein graft (p <0.001). Baseline characteristics of propensity - matched patients based on sequential venous grafts bmi: body mass index; copd: chronic obstructive pulmonary disease; iabp: intra - aortic ballon pump; lima: left internal mammary artery; lvedd: left ventricular end diastolic diameter; nyha: new york heart association; pci: percutaneous coronary intervention; rwma: regional wall motion abnormity; sd: standard deviation . Procedural characteristics of propensity - matched patients based on sequential venous grafts * values for svg and ivg respectively . Ivg: individual vein graft; svg: sequential vein graft; vg: vein graft . No significant differences was present in in - hospital composite endpoints, with 14 (11.0%) total events in individual venous grafts group versus 18 (14.2%) in sequential venous grafts group (p = 0.45). Also, there were no significant differences between individual venous grafts and sequential venous grafts group with regard to the individual outcome of in - hospital mortality (0.8% vs. 0.0%, p = 1.00), mi (0.8% vs. 0.8%, p = 1.00), stroke (0.8% vs. 0.8%, p = 1.00), iabp assistance (0.0% vs. 0.8%, p = 1.00) and prolonged ventilations (10.2% vs. 13.4%, p = 0.44). In - hospital outcomes of propensity - matched patients based on sequential venous grafts iabp: intra - aortic ballon pump . Composite in - hospital outcome includes in - hospital mortality, myocardial infarction, stroke; iabp requirement and prolonged ventilation . Major adverse cardiac events occurred in six (4.7%) patients (two deaths, two mis and two pcis) in individual venous grafts group and two (1.6%) patients (two mis) in sequential venous grafts group . The kaplan - meier mace - free survival estimates at about four years were 92.5% and 97.3% in the individual and sequential venous grafts group respectively (p = 0.36; figure 1a). The hr for sequential vein bypass grafting was estimated at 0.21 (95% ci, 0.031.85, p = 0.16). The survival rates free of angina recurrence at about 4 years was 80.9% in patients receiving individual venous grafts only versus 85.5% in patients receiving sequential venous grafts (p = 0.48; figure 1b), with hr for sequential vein bypass grafting at 0.70 (95% ci, 0.271.83, p = 0.47). Event - free kaplan - meier estimates for individual venous grafts group and sequential venous grafts group respectivly . Shown are percent survival free from mace (a) and survival free of angina recurrence (b). This retrospective analysis of 127 propensity - matched pair of patients is performed in an attempt to assess the impact of sequential vein bypass grafting on both in - hospital and mid - term clinical outcomes in the setting that there was limited and inconsistent data concerning clinical outcomes of sequential vein bypass grafting and that published reports on this subject were conducted mostly in patients undergoing on - pump cabg and failed to exclude the impact of different level of surgical expertise with sequential grafting technique . The present study suggests that sequential vein bypass grafting technique was not independently associated with increased risk of composite or individual outcomes of in - hospital mortality, mi, stroke, iabp assistance and prolonged ventilation in patients undergoing off - pump cabg . Moreover, mid - term maces and angina recurrence was also comparable between sequential venous grafts group and individual venous grafts group . Sequential grafting was introduced by flemma et al . In 1970s . A single vein used as sequential graft can revascularize whole heart as a snake graft . The key concept of sequential grafts is decreased total resistance to graft flow by having more than one target per segment . Indeed, the resistance in a sequential circulation is decreased with two or more distal anastomoses because the size of vascular bed is increased so that flow increase . This notion was supported by our observation that blood flow in sequential vein graft was significantly increased compared with individual vein graft, which was in line with the results reported by nordgaard et al . Through this higher flow the main bypass trunk stays open and may assist in maintaining patency of side - to - side anastomoses to arteries with a poor runoff . One recent meta - analysis by li et al ., which included 12 studies and compared 6838 sequential grafts towards 3285 individual vein grafts, reported that the mid - term and long - term risk of occlusion in sequential grafts was lower than that in individual grafts (risk ratio 0.67, 95% ci 0.600.74, p <0.0001). Studies regarding the effect of sequential vein bypass grafting on clinical outcome in on - pump cabg had yielded inconsistence results . Early favorable results from the study by bigelow et al . Involving 130 patients, revealed benefits of better survival with sequential vein bypass grafting, compared to multiple individual grafts . Recently reported the results of an observational study to evaluate the effect of sequential vein bypass grafting on early and long - term clinical outcomes in 452 propensity matched patients . They observed that sequential vein bypass grafting group was even comparable to total arterial revascularization group in in - hospital mortality, long - term survival, repeat pci, mi and even reappearance of angina during a mean follow - up of 14 4 years . Despite purported benefits, concerns have centered on the dependence of multiple distal anastomoses on a common inflow with the possibility of catastrophic consequences in the event of a proximal occlusion, thus leading to the patient's life at stake . After having followed up 428 patients in 15 years following a coronary revascularization procedure, van brussel et al . Found although more complete revascularization was obtained in patients with sequential vein grafts only, more events (death, mi and reintervention), especially mi, occurred in these patients than in patients with individual vein grafts only . Likewise, mehta et al . Recently reported in a subanalysis of the prevent iv trial that patients with sequential vein bypass grafting were more likely to have grafts failure, a trend towards higher death, acute mi or repeated revascularization in the 5 years following surgery . All those studies concerning the effect of sequential vein bypass grafting on clinical outcomes were done using patients who had on - pump cabg . Off - pump cabg is now gaining increasing recognition, especially in asian countries, which account for at least 60% of all the cabg operations . However, the impact of sequential vein bypass grafting is seldom investigated in off - pump cabg . Compared to on - pump cabg, sequential vein bypass grafting in off - pump cabg is technically more demanding due to its increased difficulty of conduit manipulation and complexity of certain distal anastomoses during beating heart surgery . Such feature of off - pump cabg may exert great impact on the quality of constructing the anastomoses and eventually on clinical outcomes . The present study shows that compared to individual vein bypass grafting, sequential vein bypass grafting was not associated with an increase of either in - hospital or mid - term adverse events in patients undergoing off - pump cabg and additionally, the hazard of proximal occlusion of a sequential grafts, leading to severely adverse cardiac events, might be overestimated . Findings from christenson et al . Also failed to detect significant survival benefits with sequential vein bypass grafting and suggested that a proximal occlusion of a sequential bypass resulted in renewed angina without infarction or sudden death in most instances . It is possible that a proximally occluded sequential vein graft will function as a large collateral vessel, provided that the terminal anastomosis is connected to a large coronary vessel with high blood flow . Similarly, our findings corresponded well to both studies described by meeter et al . And ouzounian et al ., who demonstrated in their study that sequential grafting was not found to be an independent predictor of short - term and long - term adverse events after adjusting for baseline difference in patients receiving on - pump cabg . It might appear strange that although the mid - term survival rate free from maces and angina recurrence is higher in sequential venous grafts group in the present study, the purported benefits of sequential vein bypass grafting did not necessarily translate into statistically significant difference of those clinical outcomes . A reason, as we speculated, might be that the period of follow - up of the present study may be not long enough to detect any significant group difference of mid - term outcome . Furthermore, this finding could be explained in part by the fact that incomplete sequentialization of distal venous anastomosis (69%, 255 sequential venous anastomoses in 369 distal venous anastomoses) in sequential venous grafts group might have also diluted the beneficial effect of sequential vein bypass grafting . The results of this study support the use of either grafting technique for venous conduits by surgeons at their discretion . Sequential grafting should be preferably considered in the following clinical scenarios: patients with athero - aortic disease with the desire to limit manipulation of the proximal aorta; patients with limited length of graft material and small target vessels and patients with special requirement for reducing hypoperfusion time of certain organs during operation; younger patients with the need for total arterial grafting . However, further studies are needed to validate the safety of sequential grafting technique for total arterial revascularization . One major limitation was that it was a retrospective study which possesses limitations due to the inherent biases that exist in nonrandomized, unblinded studies, thus limiting the generalizability of our conclusions . Although propensity matching method, which is by far the best method of comparison in observational settings, was used, the unknown variables that affect the outcomes of interest could not be fully eliminated . Moreover, the sample size in our study was relatively small because we only included patients operated by one surgical team to minimize the surgical technique and expertise viability which probably was among the major determinants of graft patency or clinical outcomes . Finally, direct assessment of graft patency by means of angiographic data or coronary computed tomography was lacking to verify whether the adverse cardiac events during follow - up were resulted from grafts failure . Therefore, a further study, especially randomized controlled trial, with increasing sample size and longer follow - up is needed to confirm our results, a large multi - center, randomized, controlled trial is needed to yield high - level evidence on this issue . However, there is, so far, no clinical trial available comparing the outcome between the sequential vein bypass grafting and individual vein bypass grafting . Compared to individual vein bypass grafting, sequential vein bypass grafting was not associated with an increase of either in - hospital or mid - term adverse events in patients undergoing off - pump cabg . However, in view of widely reported benefit of higher patency rate associated with sequential vein bypass grafting, we recommended the use of sequential vein bypass grafting when feasible.
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Bacteria isolation: bhi - t 80 agar: bhi - t 80 agar and broth were used for isolation of e. rhusiopathiae strains from tissue samples . The strains were identified and confirmed as e. rhusiopathiae by polymerase chain reaction (pcr) after gram stain and microscopic examination . All strains were isolated from cases of acute swine erysipelas causing sudden death of fattening pigs and postpartum sows in 2009 (1 strain) and 2013 (7 strains) and were named after the isolation districts and time (table 1table 1.strains used in the study and eight field strains isolated from cases of septicemia causing acute death of fattening pigs and postpartum sows in eastern chinastrainsdate isolatedarea isolatedgenbankaccession numberserotypecountry ofisolationserotype 1a (fujisawa)unknownunknownab259652.11ajapang4t10unknownunknownkj6450721aunknownsy09102727/10/2009siyangkj6450801achinanz13070101/07/2013nanzhangkj6450731achinahx13070909/07/2013hexiankj6450741achinaxc13071010/07/2013xuanchengkj6450751achinash13072323/07/2013sihongkj6450761achinayc13082020/08/2013yanchengkj6450771achinayc13082828/08/2013yanchengkj6450781achinayc13111515/11/2013yanchengkj6450791achinaa) isolates yc130820, yc130828 and yc131115 were originated from three different farms in yancheng . ). A) isolates yc130820, yc130828 and yc131115 were originated from three different farms in yancheng . G4t10 strain (serotype 1a), a widely used commercial vaccine in china, was used as a reference attenuated strain with acriflavine resistance . Serotyping: serotyping was done on all the 8 field e. rhusiopathiae strains as described previously with some modifications . Tanja opriessnig (veterinary diagnostic laboratory, college of veterinary medicine, iowa state university, ames, iowa, u.s.a . ). In brief, a pure culture was grown at 37c for 48 hr in 30 ml of bhi broth (binhe microorganism reagent, hangzhou, china) supplemented with 10% bovine serum (tianhang biological technology, deqing, china). The culture was treated with formalin solution (final concentration of 1%) (xilong chemical corporation, shantou, china), held at room temperature for 24 hr, harvested by centrifugation and washed three times in 1 pbs buffer containing 5% formalin . Washed cells were suspended in 1 ml of distilled water and autoclaved at 121c for 45 min . The supernatant was collected and delivered to dr . Analysis of the 432-bp hypervariable region in the spaa gene of different strains: primers erko-1f (5-gtgaaacaccgtattttagta-3) and erko-2r (5- ttcaagaagttcctgtagttt-3) were used to amplify the highly variable region of the spaa gene . Briefly, genomic dna of e. rhusiopathiae strains was prepared using bacterial dna kit (omega, norcross, ga, u.s.a . ). Polymerase chain reaction (pcr) was performed as described elsewhere under the following conditions with some modifications: primestar hs dna polymerase (takara, tokyo, japan) was used to perform pcr; a denaturation step at 94c for 3 min followed by 35 cycles of denaturation at 94c for 45 sec; annealing at 55c for 30 sec and extension at 72c for 30 sec; and final extension of 5 min . For each strain, pcr was performed three times, and each time, the pcr products were ligated with the pmd19-t simple vector (takara), and then, three colonies of each ligation product were sent to invitrogen (life technologies, shanghai, china) for sequencing . Pulsed - field gel electrophoresis: chromosomal dnas from the strains were digested with smai, and pfge was performed 3 times according to opriessnig et al .. electrophoresis was carried out for 23 hr at 14c and 6 v with pulse times from an initial 1.2 sec to a final 30 sec . Pfge patterns were detected by uv transillumination after staining by sybr gold nucleic acid gel stain (s11494, life technologies). A lambda ladder pfg marker (n0340s, neb, ipswitch, ma, u.s.a .) Was used as dna size standard . Acriflavine resistance test: the acriflavine resistance of all the eight strains was examined by streaking strains onto agar plates containing 0.04, 0.03, 0.02, 0.015, 0.01, 0.075, 0.005, 0.0025, 0.00125 or 0% acriflavine (aladdin, shanghai, china). The acriflavine agar plates were prepared by adding 1% acriflavine (dissolved in distilled water) to the sterilized basal medium (bhi - t 80 agar). The isolates were cultivated in 3 ml bhi - t 80 broth overnight at 37c and were then streaked onto the agar plates . The vaccine strain (g4t10) the growth on the plates was observed after incubation at 37c for 48 hr . Acriflavine resistance was determined by the highest concentration at which a strain showed almost the same growth as on the agar that did not contain acriflavine [8, 9, 11]. Attenuation of strain hx130709: strain hx130709a (derived from strain hx130709) was attenuated by 55 passages on bhi - t 80 agar containing a gradually increasing concentration (0.0025% to 0.03%) of acriflavine dye . Finally, the 432-bp partial spaa genes of all the archived strains were amplified for sequence analysis, and the virulence of each archived strain was confirmed in mice . Mouse pathogenicity tests: one hundred and sixty - five female icr mice of six - week - age were purchased from the laboratory animal centre of nantong university (nantong, china). The animals were cared for in accordance with the guide for the care and use of laboratory animals (1996, published by national academy press, nw, washington, dc, u.s.a . ). The animal experiments were approved by the institutional animal care and ethics committee of nanjing agricultural university (approval no . A total of 90 female icr mice of six - week - age were given subcutaneous injections in the right groin with 10 colony forming units (cfu)/ 0.1 ml of the isolates or the vaccine strain . A group of 10 mice served as an untreated control . Since a positive correlation between the isolate s cfu and od value had been built in advance (data not shown), the number of bacteria injected into each mouse was almost the same . Mice were observed every 12 hr to detect clinical signs of the disease for the subsequent 14 days . Another 60 female icr mice of six - week - age were given subcutaneous injections in the right groin with 10 cfu/ 0.1 ml of the isolate strain hx130709 (f0) and different passages of the attenuated strain hx130709a (f5, f10, f15, f20, f25, f30, f35, f40, f45, f50 and f55). Antimicrobial susceptibility test: each isolate was cultured in 3 ml bhi - t 80 broth overnight at 37c . Then, 0.1 ml of the bacterial culture of each isolate was dropped onto bhi - t 80 agar, after which, antibiotic susceptibility discs (binhe microorganism reagent) were put onto the bhi - t 80 agar . The diameter of the bacteriostatic circle was measured after incubation at 37c for 24 hr . The susceptibility was determined according to instructions of the clinical and laboratory standards institute (clsi). Serotyping and acriflavine resistance: all the eight e. rhusiopathiae isolates were determined to be serotype 1a (fig . 1.genetic relationship between 8 e. rhusiopathiae field isolates and 1 vaccine strain and schematic representation of 8 different pfge patterns obtained after restriction digestion with sma i. the classification and divergence of isolates were calculated by the unweighted pair group method with averages from the pfge results . At 5% divergence, 3 pfge groups (a - c) were present; group a was subgrouped into a1/a2 at 3.5% divergence . ), which was finished by the laboratory of dr . Tanja opriessnig (veterinary diagnostic laboratory, college of veterinary medicine, iowa state university). The results of the acriflavine resistant test are shown in table 2table 2.acriflavine resistance of e. rhusiopathiae field isolates and vaccine reference strainsstrainmic of acriflavine(% w / v)acriflavineresistancesy0910270.02rnz1307010.005shx1307090.0025sxc1307100.02rsh1307230.02ryc1308200.0025syc1308280.005syc1311150.0175rg4t10 (vaccine)0.02ra) resistant (r) or sensitive (s) to 0.01% acriflavine .. out of the total 8 isolates, 3 (37.5%) showed culture growth on the bhi - t 80 agar containing 0.02% acriflavine, which was identical to the growth of g4t10 strain (positive control). Genetic relationship between 8 e. rhusiopathiae field isolates and 1 vaccine strain and schematic representation of 8 different pfge patterns obtained after restriction digestion with sma i. the classification and divergence of isolates were calculated by the unweighted pair group method with averages from the pfge results . At 5% divergence, 3 pfge groups (a - c) were present; group a was subgrouped into a1/a2 at 3.5% divergence . Sequence analysis of the 432-bp hypervariable region in the spaa gene: according to nagai et al ., different nucleotide substitutions in the 432-bp hypervariable region on the spaa gene of the eight e. rhusiopathiae field strains and the japanese official challenge strain fujisawa comparing with the vaccine strain g4t10 are shown in table 3table 3.substitutions in nucleotide and amino acid in a 432-bp hypervariable region on the spaa gene of 8 e. rhusiopathiae field strains compared with the corresponding sequence of the vaccine strain g4t10substitutionpattern / strainsubstitutions (position [nucleotide], amino acid) no . Of nucleotidesubstitutionsspaa type584 (a)590 (t)609 (t)769 (a)885 (a)d> ai> ti> mi> lnspattern 1nz130701c1ayc130820c1asy091027c1apattern 2hx130709g1bsh130723g1byc131115g1bpattern 3yc130828cg2cpattern4xc130710cg2dreference strainfujisawac1a) original amino acid> substituted amino acid; ns = no amino acid mutation; g = glycine; d = aspartic acid; n = asparagine; a = alanine; i = isoleucine; t = threonine; m = methionine; l= leucine; e = glutamic acid . B) japanese official challenge strain .. according to the different substitutions, spaa can be divided into four spaa - types . No nucleotide substitutions in the 432-bp hypervariable region on the spaa gene of all the archived attenuated strains were found comparing with the parental strain hx130709 (table 4table 4.nucleotide substitutions in a 432-bp hypervariable region on the spaa gene of 11 e. rhusiopathiae strains during the attenuation compared with the corresponding sequence of the original strain hx130709concentration ofacriflavine (% w / v)number ofpassagebacteria collectedfor spaa sequencingno . Of nucleotidesubstitutionsspaa type0.0025f1-f40.00375f5-f7f50b0.005f8-f10f100b0.0125f11-f120.02f13-f27f15, f20, f250b0.0225f28-f38f30, f350b0.025f39-f45f40, f450b0.0275f46-f480.03f49-f55f50, f550breference strainhx130709f0b). A) original amino acid> substituted amino acid; ns = no amino acid mutation; g = glycine; d = aspartic acid; n = asparagine; a = alanine; i = isoleucine; t = threonine; m = methionine; l= leucine; e = glutamic acid . Pfge and data analysis: eight distinct pfge patterns with 22 to 30 dna fragment bands were produced from the genomic dna of the 8 isolate strains and 1 vaccine strain g4t10 with smai digestion (fig . 2.pfge patterns produced from 8 erysipelas field isolates and 1 vaccine strain digested with sma i. lanes: 1, g4t10; 2, nz130701; 3, hx130709; 4, xc130710; 5, sh130723; 6, yc130820; 7, yc130828; 8, sy091027; 9, yc131115; m, lambda ladder pfg marker (n0340s, neb).). Genetic relationships among the isolates were compared, and the dendogram analysis was done (fig . 3 pfge groups (a - c) were present at 5% divergence . At 3.5% divergence, most of the isolates (6/9) were within group a including the vaccine strain g4t10 . Resistance to acriflavine and spaa - types showed no specific patterns and were randomly distributed . Data analysis of the homogeneity of the pfge patterns showed that these 9 strains shared over 92.1% identity with each other . Pfge patterns produced from 8 erysipelas field isolates and 1 vaccine strain digested with sma i. lanes: 1, g4t10; 2, nz130701; 3, hx130709; 4, xc130710; 5, sh130723; 6, yc130820; 7, yc130828; 8, sy091027; 9, yc131115; m, lambda ladder pfg marker (n0340s, neb). The pfge patterns produced from the genomic dna of the 12 archived strains during the attenuation with smai digestion are shown in supplemental fig . The pfge patterns of these 12 strains were very similar with each other . A few changes of patterns occurred between strains f10 and f15, but strains f15-f55 almost shared the same pfge pattern . Based on the dendogram analysis, 2 pfge groups (a - b) were present at 5.8% divergence (fig . 3fig . 3.genetic relationship among the 12 archived e. rhusiopathiae strains during the attenuation and their pathogenicity towards mice . The classification and divergence of the strains were calculated by the unweighted pair group method with averages from the pfge results (supplemental fig . 1). Most of the strains (9/12) were within group a in which these 9 strains shared over 99.5% identity with each other . Each mouse in different groups was injected with 0.1 ml pbs and 10 cfu strains f0-f55, respectively . Mice were observed daily to detect clinical signs of the disease for the subsequent 14 days . Only 3 groups survived for the end of the experiment after receiving subcutaneous injections of 10 cfu of strains f50 and f55 and pbs, while other groups injected with 10 cfu of strains f0-f45 died within 5 days . ). Most of the strains (9/12) belonged to group a in which these 9 strains shared over 99.5% identity with each other . Genetic relationship among the 12 archived e. rhusiopathiae strains during the attenuation and their pathogenicity towards mice . The classification and divergence of the strains were calculated by the unweighted pair group method with averages from the pfge results (supplemental fig . 1). Most of the strains (9/12) were within group a in which these 9 strains shared over 99.5% identity with each other . Each mouse in different groups was injected with 0.1 ml pbs and 10 cfu strains f0-f55, respectively . Mice were observed daily to detect clinical signs of the disease for the subsequent 14 days . Only 3 groups survived for the end of the experiment after receiving subcutaneous injections of 10 cfu of strains f50 and f55 and pbs, while other groups injected with 10 cfu of strains f0-f45 died within 5 days . Pathogenicity towards mice: the results of the pathogenicity towards mice are shown in figs . 3 and 4fig . Each mouse of the untreated control group received a subcutaneous injection of 0.1 ml pbs in the right groin and was observed every 12 hr as others . Almost every mouse injected with 10 cfu e. rhusiopathiae field strains died within 72 hr, while all mice injected with 10 vaccine strain g4t10 and pbs survived for the end of the experiment .. regardless of the acriflavine resistance, pfge patterns or spaa types, all the 8 isolates and the early passages of the attenuated strains (f5, f10, f15, f20, f25, f30, f35, f40 and f45) showed high levels of virulence in mice . All mice died within 5 days after receiving subcutaneous injections of 10 cfu of the strains, while all those injected with the attenuated strains (g4t10, f50 and f55) and pbs survived for the end of the experiment . Each mouse of the untreated control group received a subcutaneous injection of 0.1 ml pbs in the right groin and was observed every 12 hr as others . Almost every mouse injected with 10 cfu e. rhusiopathiae field strains died within 72 hr, while all mice injected with 10 vaccine strain g4t10 and pbs survived for the end of the experiment . Antimicrobial susceptibility: diameters of the bacteriostatic circles of the 8 isolates, vaccine strain g4t10 and the attenuated strain hx130709a (f55) against the 15 antimicrobial agents are shown in table 5table 5.antimicrobial susceptibility testing results of 8 e. rhusiopathiae field strains, the vaccine strain g4t10 and the attenuated strain hx130709aantibioticalsusceptibility discs diameter of bacteriostatic circle (mm) no . Of resistantstrains (%) g4t10nzxcshycycycsyhxhx130701130710130723130820130828131115091027130709130709apc - g40/s42/s42/s40/s38/s39/s42/s38/s40/s54/samo40/s39/s42/s40/s39/s40/s42/s40/s43/s55/samp36/s38/s36/s34/s38/s36/s40/s34/s36/s46/scez32/s35/s35/s34/s34/s32/s36/s33/s34/s41/sctx37/s36/s36/s42/s34/s34/s38/s33/s37/s47/ssm17/s12/i6.5/r6.5/r14/i14/i6.5/r12/i6.5/r23/s4 (40)km6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r10 (100)tc26/s30/s9/r10/r27/s27/s12/r26/s14/r39/s4 (40)doxy25/i30/s11/r14/r25/i26/i18/r27/i15/r36/s4 (40)em32/s35/s33/s34/s24/s32/s30/s35/s39/s44/sroxm30/s31/s31/s30/s22/i30/s29/s27/s30/s42/slcm36/s33s11.5/r14/r6.5/r32/s9/r30/s13/r42/s5 (50)sxz6.5/r6.5/r10/r8/r6.5/r6.5/r6.5/r6.5/r6.5/r10/r10 (100)erfx36/s6.5/r16/i11/r17/i19/i12/r34/s15/i10/r4 (40)cip30/s6.5/r20/i18/i18.5/i18.5/i17/i36/s18/i16/i1 (10)a) penicillin (pc - g), amoxicillin (amo), ampicillin (amp), cefazolin (cez), cefotaxime (ctx), streptomycin (sm), kanamycin (km), tetracycline (tc), doxycycline (doxy), erythromycin (em), roxithromycin (roxm), lincomycin (lcm), sulfisoxazole (sxz), enrofloxacin (erfx) and ciprofloxacin (cip). S = susceptible; i = intermediate; r = resistance .. all the strains were highly sensitive to pc - g, amp, cez, ctx, em, roxm and amo . Half of the isolates (hx130709, xc130710, sh 130723 and yc131115) were resistant to sm, tc, doxy and lcm, and the other isolates (nz130701, yc130820, yc130828 and sy091027) were moderately resistant to these antibiotics . A) penicillin (pc - g), amoxicillin (amo), ampicillin (amp), cefazolin (cez), cefotaxime (ctx), streptomycin (sm), kanamycin (km), tetracycline (tc), doxycycline (doxy), erythromycin (em), roxithromycin (roxm), lincomycin (lcm), sulfisoxazole (sxz), enrofloxacin (erfx) and ciprofloxacin (cip). Compared with strain hx130709, the attenuated strain hx130709a (f55) was much more sensitive to these antibiotics, except for km, sxz, erfx and cip . Swine erysipelas used to be widely spread twenty years ago in china, which was later well controlled due to the wide use of antibiotics . In the present study, all the eight e. rhusiopathiae isolates were from cases of septicemia and were determined to be serotype 1a . The acriflavine resistance has been used as one of the tools for discriminating the vaccine strain from field isolates because the live vaccine strain could grow in media containing at least 0.02% acriflavine, while the field strains are usually sensitive to acriflavine . Some acriflavine - resistant e. rhusiopathiae strains have been isolated from slaughter pigs affected by chronic arthritis [8, 9, 14]. It s worth mentioning that among the eight e. rhusiopathiae strains isolated from septicemia cases in this study, 3 (37.5%) virulent isolates (sy091027, xc130710 and sh130723) showed culture growth on the bhi - t 80 agar containing 0.02% acriflavine as the vaccine strain g4t10 did . Proved that spaa plays an important role in enhancing the virulence of e. rhusiopathiae strains . A method for discrimination between a japanese vaccine strain and the field strains was developed based on the nucleotide sequencing of a 432-bp hypervariable region in the spaa gene . In this study, spaa gene analysis showed that no mutations occurred among different passages of strain hx130709a comparing with the parental strain hx130709, which further confirmed that the virulence of serotype 1a e. rhusiopathiae strains isolated from acute swine erysipelas outbreaks in eastern china is unrelated with some point mutations in the hypervariable region of the spaa gene . Prevalence of met-203 type spaa variant in e. rhusiopathiae isolates is increasing in japan . Interestingly, four of the eight isolates in our study belonged to met-203 type, and the other four field isolates belonged to ala-195 type (supplemental table 1). A further study should be carried out to investigate the prevalence of met-203 type spaa variant in e. rhusiopathiae isolates in china . Based on the dendogram analysis, the nine strains shared over 92.1% identity with each other, showing no correlations with spaa - type and resistance to acriflavine (fig . Results of the drug susceptibility test showed that all the strains were highly sensitive to a variety of antibiotics, such as pc - g, amp, cez, ctx, em, roxm and amo, and also resistant to km and sxz, which well explained why swine erysipelas was easy to control with the use of antibiotics . However, nearly half of the isolates showed resistance to lcm, sm, tc, doxy, sxz and erfx, because these antibiotics were widely added in the pig feeds in china . With the emergence of new types of e. rhusiopathiae strains which are resistant to the widely used antibiotics, vaccines seem to be a better choice in controlling swine erysipelas however, attenuated vaccine strains carry the risk of regaining their virulence thus, posing a hazard to susceptible swine, and the inactivated vaccines are rarely used because of the higher vaccination cost . So, it is extremely urgent to develop more effective and safer subunit or gene engineering vaccines with virulence factor . However, the attenuation mechanism of e. rhusiopathiae strains remains unknown . Though spaa gene analysis showed that no mutations occurred during the attenuation, the mutations probably occurred in different important points of genome . So, all the archived strains (f5, f10, f15, f20, f25, f30, f35, f40, f45, f50 and f55) and the original strain hx130709 (f0) were analyzed using pfge to detect whether any genome changes occurred during the attenuation . The pfge patterns of the above 12 strains were very similar with each other (supplemental fig . 1). A few changes of the pfge patterns occurred between strains f10 and f15, but strains f15-f55 shared over 99.5% identity with each other (fig . Surprisingly, though strains f15-f55 shared almost the same pfge patterns, only strains f50 and f55 were avirulent (fig . 3). This indicates the attenuation occurred between f46 and f50 and the mutations of smai restriction enzyme cutting site in the genome of the stains did not lead to any change of the virulence . It s possible that the mutations in the genome which directly lead to virulence loss cannot be detected by pfge with smai digestion . In this case, comparing the entire genome of wild strain and the attenuated strain may be a good way to understand the attenuation mechanism . But, there is also another possibility that the strain virulence was decreased during the attenuation mainly by phenotype changes instead of genotype changes . In this study, we conclude that the acute swine erysipelas outbreaks in eastern china were caused by serotype 1a e. rhusiopathiae strains with different biochemical characteristics, and the virulence of serotype 1a e. rhusiopathiae strains is unrelated with some point mutations in hypervariable region of the spaa gene . Meanwhile, the attenuated strain hx130709a was obtained from a highly virulent clinical strain hx130709 after 55 passages on agar plates containing acriflavine . Also, its virulence had been confirmed in mice, which lays the foundation for a further study of virulence factor of e. rhusiopathiae with proteomics and transcriptomic technologies.
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Despite significant advances in medical science and health care, it has been impossible to eliminate tb as a public health concern . Several factors have proven advantageous to the survival of mycobacterium tuberculosis (mtb) in humans, including population growth in urban environments and the resulting close living conditions; increasing numbers of persons co - infected with hiv who are more susceptible to developing tb; and increasing numbers of drug - resistant tb cases against which standard drug regimens are failing . It is noteworthy that as much as one - third of the global population is estimated to harbor mtb and may serve as a reservoir for active disease . Initially intensified by hiv / aids in countries where both diseases are endemic, the epidemic now takes close to two million lives each year . Decades of chemotherapy under often less than optimal treatment conditions have led to the development of drug - resistant tb that complicates cure . In countries with high rates of transmission and hiv co - infection, drug - resistant mtb strains now account for an increasing portion of new tb cases . The global plan to stop tb 20112015,' issued by the stop tb partnership (housed at the world health organization (who)), has set ambitious goals to halve tb prevalence and death rates by 2015, and to eliminate tb by 2050 . (elimination is defined as less than one case of tb per one million population per year .) This plan is also tied to the united nation's millennium development goals . To accomplish these milestones, scientific and biomedical innovations such as those supported by the national institute of allergy and infectious diseases (niaid) improvements in existing tb care programs alone are no longer sufficient . Effectively translating biomedical research into improved patient care and new therapeutics, preventive measures, and diagnostics requires coordination among all global tb partners . Key funders, researchers and supporters of global tb research and development have recently established several frameworks for research and program implementation . For instance, the 2011 who / stop tb partnership's an international roadmap for tuberculosis research' and priorities in operational research to improve tuberculosis care and control' create an international framework to which the research agendas of major funders and supporters, as well as biomedical science as a whole can be mapped to assure that global approaches are coordinated and key gaps identified . Niaid, part of the us national institutes of health (nih), plays a critical leadership role in tb biomedical research, supporting scientists in building a solid foundation of knowledge to advance innovations in tb . Niaid's research agenda complements efforts of other global funders, other us federal agencies, as well as public research agenda for multidrug - resistant and extensively drug - resistant tuberculosis' which outlines critical gaps in knowledge about the emergence, detection, prevention and cure of drug - resistant tb . The bill and melinda gates strategy overview: tuberculosis' in 2009, enumerating goals for intensive research and development and implementation of new products, improved use of existing tools, and advocacy for funding and political commitment to improve global tb care . Most recently, the journal tuberculosis published the 2012 tuberculosis vaccines: a strategic blueprint for the next decade,' which focuses on biomedical, product development and operational research needs that must be addressed to successfully transition vaccine candidates to the market and into public health programs . The interconnectedness and complementarity of these selected research agendas highlights the intersection between research and programmatic efforts that is crucial for closing the gaps in tb diagnosis, prevention, and treatment, and is a theme woven throughout this commentary . Although tb has been a topic of much discussion in medical texts since the nineteenth century, significant and focused biomedical research on this disease only truly commenced in the 1990s . Many hallmarks of the disease were well documented decades or even centuries ago, but once antibiotics became available, tb was considered a manageable disease . With the reduction of tb deaths in industrialized countries came diminished interest in tb research, leaving many critical questions unanswered about its pathology, pathogenesis, epidemiology and transmission . Over the past decade, however, it became apparent that existing public health strategies and healthcare tools were no longer sufficient to contain the modern global tb epidemic, and new tools would be needed . Unquestionably, a solid understanding of the science behind the disease is a critical part of developing these tools . We reached an important milestone in public health when the who report global tuberculosis control 2011' identified biomedical research as a key component of the fight against tb, making it clear that not only new drugs, better vaccines and improved diagnostics, but also innovative ways of combining these modalities will be necessary to reduce new cases at a rate that would drive tb into elimination . Fundamental basic research will be needed to understand the detailed dynamics of host / pathogen interaction, and how this guides transmission, pathogenesis and disease resolution . For instance, it is not currently understood to what extent susceptibility to infection and disease is affected by host or pathogen genetics . Gaining a better understanding of whether and how genetic diversity among mtb strains influences their ability to infect humans and cause disease will be critical in tracking, prioritizing and intervening in tb outbreaks . Furthermore, this understanding may allow researchers to focus on strains of greatest concern when developing new drug and vaccine candidates, as well as diagnostics that can accurately differentiate tb strains . New strategies for product development in these areas can no longer be limited to separate programs; rather, in order to deliver the greatest benefit to patients, the interconnectedness of host - directed interventions (traditionally vaccines) and pathogen - directed interventions (traditionally drugs) must be explored and developed . For example, adding immune supportive vaccines to drug treatment for tb may improve effectiveness, shorten duration and ultimately improve tolerability of treatment . Conversely, vaccines combined with chemoprevention of limited duration may prove effective in a broader range of individuals infected with mtb . Guidance for discovery, development and clinical testing of new drugs, vaccines and diagnostics has been discussed at length in the recent scientific literature . However, in order to establish a robust and continuous pipeline of new products, appropriate targets for intervention at several stages in the pathogenicity cycle have to be defined in both the microbe and host . Active pulmonary tb disease, the focus of many tb control programs, is also the focus of many research studies on new interventions . Tb stages that are characterized by low numbers of bacteria and limited clinical symptoms, however, are also important and need to be investigated . To eliminate a transmissible respiratory disease as a public health concern, we must understand and intervene in the processes leading to active disease and address stable cure with the eradication of bacteria from patients . Prevention of tb is a multifaceted and complex issue that requires a deep understanding of several key issues . Of particular importance are the molecular and immunologic mechanisms of transmission and infection and the dynamics of latent (asymptomatic) and subclinical disease that precede the most transmissible form of tb . Quickly and accurately identifying the presence of mtb in people with both latent and active tb is a critical first step to prevention of additional cases . Recognizing that patients are often afflicted by more than one disease, it is important to decipher how active pulmonary and other forms of tb develop in patients with healthy immune systems as well as those who are immunocompromised . Many individuals who are exposed to mtb do not get infected, and only a fraction of those infected develop active disease . The factors that govern infection and progression to active disease are not well understood, largely because the tools needed to study these phenomena are limited . Clinical diagnosis of infected persons is currently only possible through indirect tools such as the purified protein derivative skin test or blood tests indicating prior exposure to mycobacterial antigens . These tools do not indicate when exposure happened or whether individuals will be able to control the infection or will go on to develop tb . To study these processes, niaid - supported scientists are developing tools that allow the detection of small numbers of mycobacteria in human specimens by interrogating the human immune system . By looking at how human immune cells respond to mtb antigens, they are hoping to see whether the continued presence of mtb can be differentiated from instances where infection with mtb has been successfully cleared either by the immune system or through chemotherapy . These studies, once coupled with investigations about the growth and location of mtb in infected individuals over time, may provide researchers with biomarkers that can differentiate bacterial presence and host responses in those who remain healthy compared with those who develop disease . It may appear logical to argue that every person that may be infected with mtb should receive preventive chemotherapy . Tb therapy, however, requires many months to complete and includes the potential for complications and serious side effects . The ultimate goal, rather, is to determine which persons with latent, asymptomatic infection are most likely to develop active tb disease, then provide chemotherapy or vaccines to prevent them from becoming ill and transmitting the disease to others . To assure that chemotherapeutic or preventive strategies are available for persons at greatest risk, these strategies need to be closely coupled with adequate diagnostic tests that clearly differentiate mild or subclinical active disease from latent infection with a low chance of progression . This differentiation is particularly important, because chemopreventive drugs and regimens do not adequately treat active disease and may result in the development of drug resistance . The success of current chemopreventive strategies is measured by its ability to reduce the number of new cases of tb within 12 years of treatment . However, it is not known whether preventive chemotherapeutic regimens are able to eradicate mtb from infected persons or whether they leave behind dormant bacteria that are able to reactivate later in life . To focus chemoprevention more directly on the elimination of dormant mtb bacteria from infected persons, researchers are trying to understand whether bacterial biochemical pathways involved in latent mtb differ from those involved in active, pulmonary tb and to develop drugs specific to each pathway . However, even with highly focused drug treatment strategies in communities with high rates of tb transmission and high susceptibility to disease, as is the case in hiv co - infected persons, treatment of latent tb does not necessarily protect from re - infection and repeated infections may require multiple rounds of treatment . In those instances, a stronger focus on immune protection through vaccines that are effective after exposure to mtb may offer longer lasting benefit for individual patients, and this is a concept that is currently being explored . The current tb vaccine, called bacille calmette guerin or bcg, is given to newborns in tb endemic countries . While bcg provides reasonable protection against childhood complications of tb, it does not provide reliable protection against pulmonary tb, the most transmissible form of the disease, in adolescents or adults . Hence, new vaccination approaches either aim to replace bcg or boost initial protection through bcg and provide immunity that lasts into adulthood . Alternatively, the immune responses generated by bcg may be considered as background immunity' for which re - priming and boosting vaccines must be developed . To properly direct the immune response, researchers are making progress in their understanding of how bcg elicits protection against childhood complications of tb, but fails to elicit long - lasting immunity against re - infection or development of disease later in life . These studies are serving as the foundation for further research into mtb antigens that could improve bcg efficacy or could be used later as boosters with protein - based vaccines or vectored constructs . These studies are also contributing to development of immune assays that measure desirable characteristics of novel vaccine candidates . Development of new vaccines against tb, whether to prevent infection or prevent disease, is a complex and difficult undertaking . Scientific studies are underway to determine how mtb avoids destruction by the human immune system and establishes latent infection . The goal of post - exposure vaccines is to identify and direct the immune system towards microbial antigens that are present during latent infection and to which the immune system would not usually respond . Coupled with the need to rapidly identify drug - resistant mtb in patient samples, different treatment options are needed to cure the various forms of drug - resistant tb . New combinations of novel antibiotic classes against which drug resistance has not yet developed are considered the critical next step to save patients' lives and limit the spread of drug - resistant tb . Several organizations and consortia are evaluating new combinations of experimental and existing tb drugs to arrive at more potent regimens . For example, the critical path to tb drug regimens initiative,' in collaboration with stakeholders representing the tb biomedical, clinical and product development communities, is exploring regulatory pathways for drug regimens . Currently, different combinations of first- or second - line drugs are recommended for treatment of drug - susceptible and drug - resistant tb . Clinical resistance against rifampin and isoniazid termed multidrug - resistant tb (mdr tb)indicates that these components of first - line therapy are no longer effective and that second - line therapeutics are indicated . In 2006, these xdr tb strains failed to respond to key components of first- and second - line therapeutics (rifampin, isoniazid, fluoroquinolones and any one of the injectable agents (amikacin, kanamycin or capreomycin)). Current treatment of drug - sensitive tb requires taking 4 to 5 drugs in combination for 6 months or more, while treatment for drug - resistant tb can require therapy for up to 2 years . Dramatic increases in mdr and xdr tb, and documentation of strains with resistance patterns that are not defined as mdr or xdr tb, necessitates the development of new classes of antibiotics and the rapid identification and treatment of infectious patients to curb the spread of drug - resistant tb . Persistent bacterial populations, a phenomenon occurring in many bacterial species in response to antibiotic pressure, are implicated as the cause for prolonged tb treatment and various approaches are being pursued to address this problem . For the development of new classes of antibiotics, niaid - supported researchers and others are determining what specific biochemical pathways are most vulnerable to drug intervention in non - replicating, persisting microbes and whether treatment can be shortened when drugs are combined in innovative ways . Studying low numbers of bacteria in host tissues or under metabolic conditions that induce non - replicating states is a requirement to understand the physiological processes that bacteria utilize to avoid antibiotic killing . A different approach that is starting to generate interest in the research community is to treat tb disease in a multifactorial way, combining antimicrobial drugs with engineered vaccines or immune stimulatory molecules to engage the host immune system in the clearance of bacteria or the prevention of persistent populations . Researchers are studying the impact of antibiotics on the host immune response and whether vaccine candidates can expedite bacterial clearance . Animal models are of particular importance in these studies since even low numbers of bacteria can be measured in homogenized animal tissues, and give a good initial estimate of a drug's effectiveness against mtb . It is expected that a combination of novel - acting drugs and an engaged host immune system may hold the key to rapid universal treatment of tb irrespective of its drug susceptibility status . Until these new treatment options are available, however, the most urgent needs are to increase treatment options for patients with drug - resistant tb and to focus on the development of molecular diagnostics that rapidly indicate whether and what type of drug resistance is present . However, many scientific findings that do not have direct potential to be translated into drugs, vaccines, or diagnostics can be invaluable for studying new interventions in animals and humans . As few new drugs and no new vaccines have been licensed for several decades, evaluation of products through clinical trials is being done at the same time that assays to support regulatory processes are being developed and evaluated . Improving standard of care for tb is particularly challenging since interventions exist against which new approaches will have to be compared . Although generally effective against tb, a better understanding of the efficacy of first- and second - line regimens, such as the bangladesh regimen,' is needed to improve the standard of care and select the most appropriate comparator regimen for licensure of additive or replacement drugs, or completely new regimens . For example, individual and population data on pharmacokinetics / pharmacodynamics and drug drug interactions, particularly with hiv - positive patients utilizing antiretroviral therapy, will help develop models to improve our understanding of the efficacy and failure of current first- and second - line drugs when used under treatment program conditions . In preparation for this, scientists are beginning to accumulate pharmacokinetic / pharmacodynamic information on several new drugs in animal and clinical studies . These data will not only contribute to baseline knowledge but may help adjust or set current standards for optimal dose . In addition, niaid has recently made available their hiv / aids clinical trial networks to assist in the complex testing that will be required to obtain and accumulate clinical data . As previously mentioned, bcg, while not effective at preventing adult or adolescent pulmonary tb, nevertheless provides benefit to infants and also offers reasonable protection against leprosy in many countries . Therefore, trials replacing bcg for the purpose of improving protection later in life must be preceded by sufficient efficacy studies indicating that a new vaccine or vaccine / adjuvant combination will continue to provide protection for these groups of individuals while contributing to increased efficacy for preventing adult pulmonary tb . Similar to datasets needed to develop new treatment regimens and schedules, controlled data on baseline efficacy and immunogenicity of bcg needs to be generated, particularly since performance of bcg in infants has not been rigorously assessed to date . A critical research need informing both vaccine studies and clinical care alike is the identification of clear clinical definitions of mtb infection and tb disease, particularly in infants and persons with compromised immune function . These definitions will facilitate inclusion of appropriate individuals in clinical trials and assessment of success (protection) or failure (disease) of vaccine candidates . Combined with thorough characterization of immune responses pre- and post - vaccination, case definitions are also helpful in defining what preclinical parameters should be assessed in animal models to select the most promising vaccine candidates / adjuvants and schedules . Standardized immune assays and reagents are currently being developed and will be evaluated in upcoming clinical trials . To assist in the development of preclinical and clinical tools, niaid provides standardized reagents and vaccine and drug testing services in animal models of tb, and also provides non - clinical development and manufacturing resources to help complete data packages needed for regulatory approval of clinical trials . Tools such as preclinical animal and ex vivo models need to be developed in parallel with product development efforts in order to assist product developers to: demonstrate that candidate drugs and vaccines warrant clinical investigation, identify patient populations that are most appropriate to be included in these trials, and complete trials in the most efficient manner . Many of the fundamental and basic biomedical research studies currently underway have the potential to contribute to these goals . Antigens recognized by the human and animal immune system may have potential as vaccine candidates and also as biomarkers to indicate the presence of live bacteria . Biomarkers may be integrated into diagnostic tools to identify relevant patient populations, and are also useful in clinical trials to follow early response to therapy . Rapid identification of mtb strains with different disease - causing potential may help curb epidemics and also provide opportunities for use in animal studies to ensure that vaccines and drugs are effective against diverse clades of mtb present in endemic areas . All approaches focused on studying mtb during what may be called paucibacillary' stages of infection or disease, defined as asymptomatic infection or subclinical disease without pathological lesions, are complicated by the lack of access to human specimens that contain sufficient bacteria for study . Animal models have been used to induce paucibacillary stages of disease through pre - treatment with antibiotics, but their relevance to human disease remains unclear . Historically, much of tb research has been driven by hypotheses generated in mouse, guinea pig, or non - human primate models without much opportunity to validate these hypotheses in humans . Through improvements in clinical study capacity in tb endemic countries, biomarkers' or biosignatures,' which are molecular markers that characterize various stages of infection and disease . Critical to the success of identifying relevant markers of tb is access to clinical samples from diverse, longitudinal studies in human cohorts . It is important to gain an appreciation of the diversity of human tb in various high burden countries as it is influenced by co - infections and comorbidities, and to understand the diversity of genetic variants of mtb and how they influence transmission, infection or the likelihood of progressing to active tb . A systems biology analysis of these markers is likely to shed light on the interconnectedness of host and microbial pathways and identify new early microbial and host markers that will lead to effective diagnostics, drugs and vaccines . The level of innovation that will be required to truly transform the way we develop new interventions in tb necessitates that researchers, who are currently operating in separate parallel tracks, join forces to apply cross cutting science to these difficult issues . For instance, understanding molecular mechanisms driving host pathogen interactions will aid in developing improved diagnostics, vaccines and drugs, and in identifying early markers of immune protection and treatment response . Biomarker discovery projects that focus on diagnosis, response to therapy, or immune protection will provide important information for product development . However, to add to our understanding of the complexity of tb, these studies and markers must be analyzed in combination . For this, bioinformatics tools and algorithms to create systems approaches are essential . In addition, comparative studies to define commonalities and differences in tb globally will be a crucial but difficult and resource - intensive undertaking . In an era of uncertain global economics, close collaboration between funding organizations, scientists, and other stakeholders will be required to utilize existing resources effectively, minimize duplication of effort and foster creative science . Valuable specimens from human volunteers must be analyzed and preserved in a way that provides maximum benefit for the overall understanding of tb and the translation of research findings to improve overall care and control of this disease . This will, however, require close collaboration between basic researchers, clinicians, and animal modelers in order to arrive at the key public health and medical questions in tb and how biomedical and particularly basic research can be applied to solve them.
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Most infections with t. gondii occur by ingestion of food or water contaminated with oocysts shed by cats [3, 4] and eating undercooked or raw meat containing tissue cysts [3, 5]. However, some t. gondii - infected individuals develop chorioretinitis that may progress to blindness . Immunocompromised patients infected with t. gondii may develop severe neurological disease [7, 8]. In addition, primary infections with t. gondii during pregnancy may lead to congenital disease [3, 8]. Infections with t. gondii may manifest in the heart in humans [9 - 13] and animals [14 - 17] with myocarditis [18 - 20], pericarditis with myocarditis [21, 22], and acute heart failure [23, 24]. Patients with t. gondii myocarditis may present with pericardial effusion, constrictive pericarditis, congestive heart failure, and arrhythmias . The seroepidemiology of infection with t. gondii in patients suffering from heart diseases has been poorly studied . We are not aware of any data about the epidemiology of t. gondii infection in these patients in mexico . Therefore, we determined the association between t. gondii exposure and patients with heart disease attending in a public hospital in northern mexico, and the association of seropositivity to t. gondii with socio - demographic, behavioral, and clinical characteristics of these patients . Through a case - control study, we enrolled 400 patients suffering from heart diseases attending in a public hospital in durango city, mexico and 400 control subjects without heart diseases of the same city . Inclusion criteria for the cases were: 1) inpatients with heart disease attending in the cardiology department at the general hospital of the secretary of health in durango city; 2) aged 11 years and older; and 3) that voluntarily accepted to participate . Inclusion criteria for the control subjects were: 1) people without heart diseases from the general population of durango city; and 2) who voluntarily accepted to participate in the study . Patients included 156 (39%) males and 244 (61%) females with a mean age of 58.87 14.59 years (range 11 - 93 years). Controls included 156 males and 244 females with a mean age of 58.76 14.54 years (range 9 - 91). This study was approved by the ethical committee of the general hospital of the secretary of health in durango city, mexico . A written informed consent was obtained from all participants and from the next of kin of minor participants . For calculation of the sample size, we used a 95% confidence level, a power of 80%, a 1:1 proportion of cases and controls, a reference seroprevalence of 6.1% as the expected frequency of exposure in controls, and an odds ratio (or) of 2.1 . The socio - demographic, clinical, and behavioral characteristics of the patients were obtained with the aid of a standardized questionnaire . Socio - demographic data included age, sex, birthplace, residence, educational level, occupation, and socioeconomic status . Clinical data included diagnosis of the heart disease, evolution time (years) of the heart disease, functional classification of the heart disease, and response to treatment . To evaluate the functional class of heart disease, we used the criteria of the new york heart association . In addition, we obtained clinical data about the presence of underlying diseases, history of lymphadenopathy, surgery, blood transfusion or transplants, presence of frequent headaches, dizziness, and impairments of memory, reflexes, hearing, and vision . Behavioral data included contact with animals, cleaning cat excrement, foreign traveling, consumption of meat (pork, beef, goat, lamb, boar, chicken, turkey, pigeon, duck, rabbit, venison, squirrel, horse, opossum, or other), frequency of meat consumption, consumption of raw or undercooked meat, unpasteurized milk, dried or processed meat (ham, sausages or chorizo), consumption of unwashed raw vegetables or fruits, untreated water, frequency of eating away from home (in restaurants or fast food outlets), contact with soil (gardening or agriculture), hand washing before eating, and type of flooring at home . Gondii igg antibody levels were expressed as international units (iu)/ml . A cut - off of 8 iu / ml was used for seropositivity . In addition, sera of patients positive for anti - t . Toxoplasma igm kit (diagnostic automation inc ., calabasas, ca, usa). The statistical analysis was performed with the software: epi info version 7 and spss version 15.0 . We used the student s t - test to compare age among cases and controls . The pearson s chi - square test and the fisher exact test (when values were small) were used for comparison of the frequencies among groups . Multivariate analysis was used to assess the association between the characteristics of the patients and the seropositivity to anti - t . . Socio - demographic and behavioral characteristics of the patients were included in the multivariate analysis if they had a p value 0.20 in the bivariate analysis . Or and 95% confidence interval (ci) were calculated by multivariate analysis with the enter method . Through a case - control study, we enrolled 400 patients suffering from heart diseases attending in a public hospital in durango city, mexico and 400 control subjects without heart diseases of the same city . Inclusion criteria for the cases were: 1) inpatients with heart disease attending in the cardiology department at the general hospital of the secretary of health in durango city; 2) aged 11 years and older; and 3) that voluntarily accepted to participate . Inclusion criteria for the control subjects were: 1) people without heart diseases from the general population of durango city; and 2) who voluntarily accepted to participate in the study . Patients included 156 (39%) males and 244 (61%) females with a mean age of 58.87 14.59 years (range 11 - 93 years). Controls included 156 males and 244 females with a mean age of 58.76 14.54 years (range 9 - 91). This study was approved by the ethical committee of the general hospital of the secretary of health in durango city, mexico . A written informed consent was obtained from all participants and from the next of kin of minor participants . For calculation of the sample size, we used a 95% confidence level, a power of 80%, a 1:1 proportion of cases and controls, a reference seroprevalence of 6.1% as the expected frequency of exposure in controls, and an odds ratio (or) of 2.1 . The socio - demographic, clinical, and behavioral characteristics of the patients were obtained with the aid of a standardized questionnaire . Socio - demographic data included age, sex, birthplace, residence, educational level, occupation, and socioeconomic status . Clinical data included diagnosis of the heart disease, evolution time (years) of the heart disease, functional classification of the heart disease, and response to treatment . To evaluate the functional class of heart disease in addition, we obtained clinical data about the presence of underlying diseases, history of lymphadenopathy, surgery, blood transfusion or transplants, presence of frequent headaches, dizziness, and impairments of memory, reflexes, hearing, and vision . Behavioral data included contact with animals, cleaning cat excrement, foreign traveling, consumption of meat (pork, beef, goat, lamb, boar, chicken, turkey, pigeon, duck, rabbit, venison, squirrel, horse, opossum, or other), frequency of meat consumption, consumption of raw or undercooked meat, unpasteurized milk, dried or processed meat (ham, sausages or chorizo), consumption of unwashed raw vegetables or fruits, untreated water, frequency of eating away from home (in restaurants or fast food outlets), contact with soil (gardening or agriculture), hand washing before eating, and type of flooring at home . Toxoplasma igm kit (diagnostic automation inc ., calabasas, ca, usa). The statistical analysis was performed with the software: epi info version 7 and spss version 15.0 . We used the student s t - test to compare age among cases and controls . The pearson s chi - square test and the fisher exact test (when values were small) were used for comparison of the frequencies among groups . Multivariate analysis was used to assess the association between the characteristics of the patients and the seropositivity to anti - t . Socio - demographic and behavioral characteristics of the patients were included in the multivariate analysis if they had a p value 0.20 in the bivariate analysis . Or and 95% confidence interval (ci) were calculated by multivariate analysis with the enter method . Anti - t . Gondii igg antibodies were detected in 55 (13.8%) of 400 patients with heart diseases and in 32 (8.0%) of 400 controls . Gondii igg antibodies was significantly higher in cases than in controls (or = 1.83; 95% ci: 1.15 - 2.90; p = 0.01). Gondii igg levels (> 150 iu / ml) were found in 28 (50.9%) of the 55 positive cases and in 14 (43.8%) of the 32 positive controls (p = 0.51). Gondii igm antibodies were found in 13 (23.6%) of the 55 anti - t . Gondii igg positive patients and in 19 (59.4%) of 32 anti - t . Gondii igm antibodies was significantly lower in cases than in controls (or = 0.21; 95% ci: 0.08 - 0.54; p = 0.0008). General socio - demographic characteristics of the 400 patients studied and their correlation with t. gondii seroprevalence are shown in table 1 . Seroprevalence of t. gondii infection was not associated with age, sex, residence, educational level, occupation or socioeconomic level of the patients by bivariate analysis . In contrast, patients born out of durango state had a significantly higher seroprevalence of t. gondii infection than patients born in durango state (p = 0.008). Agriculture, business, employee, cattle raising, factory worker, professional, other . Showed p values 0.20 by bivariate analysis . Whereas other diagnoses of heart disease, functional stages of heart disease, response to treatment, presence of underlying diseases, history of lymphadenopathy, surgery or transplants, presence of frequent headaches, dizziness, and impairments of memory, reflexes, hearing, and vision showed p values> 0.20 by bivariate analysis . A selection of clinical characteristics of the patients and their correlation with t. gondii seroprevalence is shown in table 2 . In female patients, seropositivity to t. gondii did not correlate with obstetric history . Among the behavioral characteristics, a number of variables showed p values 0.20 in the bivariate analysis including national trips (p = 0.12), consumption of meat from squirrel (p = 0.08), opossum (p = 0.05), armadillo (p = 0.18), iguana (p = 0.14), and snake (p = 0.19), degree of meat cooking (p = 0.05), consumption of unwashed raw fruits (p = 0.06), frequency of eating out of home (p = 0.07), frequency of meat consumption (p = 0.16), and alcohol consumption (p = 0.02). A selection of behavioral characteristics of the patients and their correlation with t. gondii seroprevalence are shown in table 3 . Other behavioral characteristics of patients including contact with cats, cleaning cat excrement, raising animals, consumption of meat other than those from squirrel, opossum, armadillo, iguana and snake, consumption of unpasteurized milk, processed meat, unwashed raw vegetables, and untreated water, and contact with soil showed p values> 0.20 in the bivariate analysis . Further analysis by using logistic regression showed that t. gondii exposure was positively associated with being born out of durango state (or = 2.93; 95% ci: 1.40 - 6.13; p = 0.004), and with consumption of alcohol (or = 2.04; 95% ci: 1.01 - 4.12; p = 0.04) (table 4). The present study was performed to investigate the association of t. gondii infection with patients suffering from heart diseases attending in a public hospital in durango city, mexico and to determine the correlates of infection in these patients . This seroprevalence is higher than the 8.0% seroprevalence found in controls, and the 6.1% seroprevalence of t. gondii infection reported in the general population in durango city . Results indicate that patients with heart diseases represent a risk group for t. gondii infection . Seroprevalence of t. gondii infection increases with age in our region [25, 27]. Therefore, we matched cases and controls by age and this strategy allowed us to evaluate properly the association between t. gondii exposure and patients with heart disease . It is hypothesized that patients with t. gondii infection have a higher risk for heart disease caused by the presence of cysts in the heart muscle . The interaction of t. gondii within skeletal muscle cells has been nicely described recently . However, to the best of our knowledge, the interaction of t. gondii within heart muscle cells has not been studied . It is unclear how t. gondii may affect the function of heart muscle . With respect to igm seropositivity to t. gondii, it is remarkable that seroprevalence of anti - t . This finding suggests that heart disease might be related with a chronic rather than a recent infection with t. gondii . However, the high frequency of igm antibodies should be interpreted with caution since igm elisa kits have a high rate of false positive results . Seroprevalence of t. gondii infection varied among heart diseases . However, this difference was not statistically significant . This might be due to a small sample size of some subgroups of specific diagnosis . Logistic regression showed that seropositivity was positively associated with being born out of durango state, and with consumption of alcohol . The characteristic born out of durango state has been associated with t. gondii seropositivity in some previous epidemiological studies in durango including adults and elderly people of the general population . On the other hand, to the best of our knowledge, this association has not been reported in alive people . It is unclear why subjects with alcohol consumption had a higher seroprevalence of t. gondii infection than those without alcohol consumption . It is uncertain whether subjects with alcohol consumption have behaviors that might favor t. gondii exposure . In a recent study of postmortem examinations in people who died suddenly in warsaw, poland, the frequency of t. gondii igg antibodies was higher in subjects with positive blood alcohol test than that in subjects with negative test . Researchers also found the highest seroprevalence of t. gondii infection among persons who died because of suicide and with positive alcohol test . Interestingly, ethanol has been involved in t. gondii invasion to and egress from the host cells, and ethanol produces a dose - dependent stimulation of microneme secretion leading to adhesion of t. gondii to host cells . Furthermore, exposure of t. gondii tachyzoites to ethanol induced conoid extrusion - an event of cell invasion - without affecting parasite viability . These facts support the association of t. gondii exposure and alcohol consumption found in the present study . Further research to elucidate the role of alcohol consumption with t. gondii exposure should be conducted . The current study was limited for the scanty number of certain heart diseases among the studied population . This limitation did not allow properly assessing differences in t. gondii seroprevalence among the diagnoses and functional stages of heart diseases . Our results indicate that t. gondii infection is associated with heart disease and suggest that heart disease might be related with a chronic infection . Results warrant for further research to determine the epidemiological impact of t. gondii exposure on heart diseases . Risk factors associated with t. gondii exposure found in the present study may help to design future prevention strategies against t. gondii infection . Our results indicate that t. gondii infection is associated with heart disease and suggest that heart disease might be related with a chronic infection . Results warrant for further research to determine the epidemiological impact of t. gondii exposure on heart diseases . Risk factors associated with t. gondii exposure found in the present study may help to design future prevention strategies against t. gondii infection.
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Graphene is a versatile two - dimensional material with applications not confined only to electronic devices . Biologists show keen interest in graphene, and various studies have been carried out to investigate its biocompatibility with cells (eg, a549 mammalian cell line on graphene and graphene oxide [go] paper, nih-3t3 mouse fibroblast cells on graphene carbon nanotube, l929 mouse fibroblast cells on graphene- and go - polyaniline and graphene - reinforced chitosan, arpe-19 retinal pigment epithelium cells on go - glucose oxidase, a549 human lung carcinoma epithelial cells on go, nano - reduced go functionalized noncovalently by amphiphilic pegylated polymer chains, and nano - go covalently pegylated on u87 mg human glioblastoma and mcf-7 human breast cancer cell lines).1 our recent work has indicated that graphene, in the form of a three - dimensional structure, may be a promising scaffold for cell growth.1 in addition to biocompatibility investigations, the use of graphene has been extended to bacterial eradication, including escherichia coli, staphylococus aureus, pseudomonas aeruginosa, and bacillus cereus.26 the most popular method employed for the synthesis of graphene is chemical oxidation of graphite.7 this method involves oxidation of graphite to go using highly oxidizing reagents and subsequently reducing go to graphene using various reductants . The advantage of this method is the formation of a large quantity of graphene in powder form (scalability), which is dispersible in both polar and nonpolar solvents by functionalizing the surface of graphene . 8 chemical oxidation of graphite is an established method using concentrated acids (sulfuric acid, nitric acid, and phosphoric acid) and highly oxidizing agents (potassium permanganate and potassium perchlorate). However, the oxidation method usually requires several steps and temperature controls for the preparation of go . A simpler method for oxidation most of the go and reduced go reported previously possessed small area and lateral dimension, which is approximately 100 m and a few hundred nanometers to a few microns, respectively.913 it is difficult to produce large - area go due to the unavoidable tearing of go sheets during the extreme oxidation condition and exfoliation process using ultrasonication . Large - area go has been reported since 2009 by tung et al14 with area <2000 m and lateral dimension of 20 m 40 m . Later, two groups reported the synthesis of ultralarge go sheets by modifying the oxidation / exfoliation of hummer s method.15,16 recently, zhao et al17 reported an efficient method to produce large - area go with an area of 7000 m and a lateral dimension of up to 100 m . However, the yield of the go obtained was about 10%, which is very low . Prior to that, luo et al18 reported the formation of go with high yield, reaching almost 100%, but the size of the go produced was merely about 2000 um . Marcano et al19 recently reported the synthesis of go using an improved hummer s method . This method introduced a relatively simple oxidation process, with temperature controlled at 50c, which was rather low compared with the previously reported temperature of 95c . Moreover, the emphasis of the method was not on producing large - area go but more on controlling the level of oxidation and degree of ordering in go . The conversion rate of graphite to go was not 100% . In order to achieve commercial value, current methods of producing go using chemical oxidation like hummer s method,20 staudeumaier s method,21 and brodie s method22 involved tedious and long experimental time . Here, the experimental time does not refer to the time required to oxidize the graphite flakes but rather the time spent by a researcher working on the experiment during the oxidation process . The long hours of mixing the reactants and cooling or heating the reactants have been reduced from 35 hours to less than 5 minutes in this work . Graphite oxidation was achieved simply by adding graphite and potassium permanganate (kmno4) into concentrated acids (containing sulfuric acid, with or without phosphoric acid) under constant stirring . The whole process was carried out without any temperature control, neither increasing nor decreasing the temperature . The mixture was stirred at room temperature for up to 3 days to achieve a high degree of oxidation . Graphite flakes (code no 3061) were purchased from asbury graphite mills, inc (asbury, nj). Sulfuric acid (h2so4, 98%), phosphoric acid (h3po4, 85%), potassium permanganate (kmno4, 99.9%), and hydrogen peroxide (h2o2, 30%), were purchased from merck (darmstadt, germany). Hydrogen chloride (hcl, 37%) was purchased from sigma - aldrich (st louis, mo). Oxidation of graphite was carried out by mixing h2so4:h3po4 (320:80 ml), graphite flakes, and kmno4 (18 g) using a magnetic stirrer . After adding all the materials slowly, the one - pot mixture was left for stirring for 3 days to allow the oxidation of graphite . Later, h2o2 solution was added to stop the oxidation process, and the color of the mixture changed to bright yellow, indicating a high oxidation level of graphite . The graphite oxide formed was washed three times with 1 m of hcl aqueous solution and repeatedly with deionized water until a ph of 45 was achieved . The washing process was carried out using simple decantation of supernatant via a centrifugation technique with a centrifugation force of 10,000 g. during the washing process with deionized water, the graphite oxide experienced exfoliation, which resulted in the thickening of the graphene solution, forming a go gel . The area, size, and morphology of the prepared go were characterized using an fei nova nanosem 400 field emission scanning electron microscope (fesem; fei, hillsboro, or). The lateral dimension and area of the graphene sheets were measured using i - solution image analysis software . Atomic force microscopy (afm) was performed on agilent 5500 (agilent technologies, inc, santa clara, ca), and a tapping mode was employed to determine the thickness of go . Go was spin - coated at 1500 rpm for 1 minute on a freshly cleaved mica substrate . The x - ray diffraction (xrd) pattern was recorded using a phillip x - ray diffractometer (philips, amsterdam, the netherlands), employing a scanning rate of 0.033s in a 2 range from 5 to 80 with cu k radiation (= 1.5418). Samples for xrd analysis were prepared by spin - coating go gel on quartz glasses . Chemical bonding was analyzed using a kratos axis ultra dld x - ray photoelectron spectrometer . Graphite flakes (code no 3061) were purchased from asbury graphite mills, inc (asbury, nj). Sulfuric acid (h2so4, 98%), phosphoric acid (h3po4, 85%), potassium permanganate (kmno4, 99.9%), and hydrogen peroxide (h2o2, 30%), were purchased from merck (darmstadt, germany). Hydrogen chloride (hcl, 37%) was purchased from sigma - aldrich (st louis, mo). Oxidation of graphite was carried out by mixing h2so4:h3po4 (320:80 ml), graphite flakes, and kmno4 (18 g) using a magnetic stirrer . After adding all the materials slowly, the one - pot mixture was left for stirring for 3 days to allow the oxidation of graphite ., h2o2 solution was added to stop the oxidation process, and the color of the mixture changed to bright yellow, indicating a high oxidation level of graphite . The graphite oxide formed was washed three times with 1 m of hcl aqueous solution and repeatedly with deionized water until a ph of 45 was achieved . The washing process was carried out using simple decantation of supernatant via a centrifugation technique with a centrifugation force of 10,000 g. during the washing process with deionized water, the graphite oxide experienced exfoliation, which resulted in the thickening of the graphene solution, forming a go gel . The area, size, and morphology of the prepared go were characterized using an fei nova nanosem 400 field emission scanning electron microscope (fesem; fei, hillsboro, or). The lateral dimension and area of the graphene sheets were measured using i - solution image analysis software . Atomic force microscopy (afm) was performed on agilent 5500 (agilent technologies, inc, santa clara, ca), and a tapping mode was employed to determine the thickness of go . Go was spin - coated at 1500 rpm for 1 minute on a freshly cleaved mica substrate . The x - ray diffraction (xrd) pattern was recorded using a phillip x - ray diffractometer (philips, amsterdam, the netherlands), employing a scanning rate of 0.033s in a 2 range from 5 to 80 with cu k radiation (= 1.5418). Samples for xrd analysis were prepared by spin - coating go gel on quartz glasses . Chemical bonding was analyzed using a kratos axis ultra dld x - ray photoelectron spectrometer . We carried out the preparation of large - area go using our simplified hummer s method . This simplified method does not involve controlling temperature during the chemical oxidation of graphite . Unlike the commonly practiced processes where an ice bath is required during the initial addition of kmno4 and heating during the oxidation stage, our simplified approach is hassle - free . The slight exothermic process, which increases the temperature to 40c50c after the addition of h2o2 solution to terminate the oxidation process, will cool down naturally in a short time . This experiment is relatively safe, and the danger of explosion is reduced significantly, in contrast to the normal route of chemical oxidation of graphite . Furthermore, the mixing and washing steps are simple and straightforward, yielding an almost 100% conversion of large - area go . To study the effect of oxidation level we used kmno4:acid (h2so4:h3po4) at the weight ratios of 1:80, 1:40, and 1:20 (hereon denoted as ks-80, ks-40, and ks-20, respectively, where the weight ratio of h2so4:h3po4 was fixed at 9:1) and the weight ratio of graphite: sulfuric acid oxidation time was fixed at 6 hours, 1 day, 2 days, and 3 days, respectively, to evaluate the oxidation level effects on go . Table 1 shows the average lateral dimension of the go produced with different ratios of kmno4:acids and oxidation time (6 hours to 3 days). After 6 hours of oxidation, go with an average lateral dimension of 18.7 m was formed in ks-20, which was larger than that of ks-40 and ks-80 . The conversion was very low, as observed from the large amount of precipitate after centrifugation for 5 minutes at 4000 rpm, indicating un exfoliated or unoxidized graphite . After 1 day of oxidation, a similar result was observed as compared with an oxidation time of 6 hours . When the oxidation time increased to 2 days, go in ks-20, ks-40, and ks-80 increased in size as compared with after 1 day of oxidation . The lack of precipitate after centrifugation suggests that ks-20 had the highest yield of go, even though the oxidation process was not yet optimized . At day 3, ks-20 achieved a 100% conversion of graphite to go, as precipitate was not found after centrifugation . The size of the go produced is large, with an average lateral dimension of 58.3 m . Figure 1a, b, and c show the fesem micrographs of graphene prepared using three different ratios of kmno4:acids (ks-80, ks-40, and ks-20) after 3 days of oxidation . The go samples were gently sonicated for 5 minutes using 80 w power of sonication before being spin - coated on sio2/si wafer and viewed under fesem . From the micrographs, it was found that the average lateral dimension of go increased as the ratio of kmno4:acid increased . Ks-20 with the highest concentration of kmno4 produced the largest go (figure 1c). However, due to the sonication effect, the average lateral dimension of graphene becomes 58.3 m . On the contrary, nansonicated go in ks-20 presents uniform sheets of go with an average lateral dimension of 95.1 m (up to 120 um) and an area of ~8000 m (figure 1d). These observations are different from the reported investigations on the effects of oxidation on the size of go . It was previously reported that the size of go reduced with a higher degree of oxidation.17,23 however, by using our proposed simplified hummer s method, go with a large lateral dimension is produced with a higher degree of oxidation . This can be explained by the fact that the initial graphite used for previous works is smaller in size as compared with that of ours, and the graphite may have overoxidized under elevated temperature (95c). As for our sample, the optimum oxidation time was 3 days (for ks-20) with the go produced possessing the largest dimension . However, after 4 days of oxidation, the go produced showed small lateral dimension in the range of <20 m (fesem micrograph not shown). Based on the fesem images, a plausible mechanism for the formation of large - area go is a complete oxidation of graphite but not overly oxidized, as it could lead to tearing of the go . The complete oxidation condition resulted in graphite oxide to be exfoliated easily in large pieces . Another contributing factor that leads to the formation of large go is that the reactions were carried out at room temperature . It has been reported that high temperature during the oxidation process reduces the size of the go produced.17 the large - area graphene for sample ks-20 after 3 days of oxidation was measured using atomic force microscopy to determine the thickness of the go produced . A typical tapping mode afm image and the corresponding height cross - section profile of the go sheets deposited on a mica substrate based on the afm height profile analysis, the thickness of the ks-20 go sample is around 1.2 nm . The result is consistent with the thickness of the characteristic single - layer go reported.17,24 ultraviolet - visible spectroscopy measurement was carried out to monitor the degree of oxidation for the graphene samples . The spectra for ks-80, ks-40, and ks-20 were recorded after 3 days of oxidation, as shown in figure 3a, with absorption peaks at 233 nm, 232 nm, and 229 nm, respectively . These peaks are due to the * transition for the c = c bonding, which is similar to the reported value in the literature.25 the peak position for ks-20 sample exhibits the lowest wavelength, indicating that the sample has a lower amount of remaining conjugation (thus requires higher energy for the electronic transition) as compared with the other two samples (ks-40 and ks-80), which is due to the higher degree of oxidation with more functional groups on the go basal plane . Also, there is a similar shoulder around 300 nm observed for all the three samples, which is attributed to n * transition of the carbonyl groups . Another observation on the ratio of the intensity of c = c bonding peak (~230 nm) to the intensity of 300 nm peak finds that the higher the degree of oxidation, the greater the ratio . This is because a more oxidized graphene basal plane yields a greater amount of isolated aromatic rings that increase the intensity of c = c bonding peak . In contrast, a less oxidized graphene basal plane gives rise to a lesser amount of isolated aromatic rings and remained in the extended conjugated aromatic rings form, and therefore the intensity of c = c bonding peak is relatively low.19 in this scenario, the peak intensity ratio for ks-20 is 3.2, and 2.1 for both ks-80 and ks-40, respectively . To the best of our knowledge, the reported ultraviolet - visible spectra for go in the literature have ratios that fall within the range of <2, in which the lateral dimension of the go produced is significantly smaller . Xrd patterns in figure 3b show a distinct diffraction peak at 10.24 for go and 26.56 for graphite flakes . The interlayer spacing values for graphite flakes and go are 0.34 nm and 0.87 nm, respectively . The distance between consecutive carbon layers was increased for go due to the introduction of oxide functional groups to the carbon basal plane via chemical oxidation reaction . This increased interlayer spacing for go is in line with the results measured from afm . In figure 4, the c1 s band of the go samples (ks-20, ks-40, and ks-80 after 3 days of oxidation) can be fitted to four deconvulated components, located at 284.3 ev, 286.6 ev, 287 ev, and 288.6 ev . These components can be assigned to the nonoxygenated ring carbon, c in c o, c in c = o, and c in c(o)o, respectively, which are separated by ~2.0 ev, 1.0 ev, 1.0 ev, and 1.0 ev.26 all the oxygenated features increased in intensity as the concentration of kmno4 increased from ks-80 to ks-20 . The components that experienced the highest degree of oxidation is the c in c = o for ks-20, as can be seen from the high - intensity peak at around 287 ev . The components are in agreement with the results in the literature for similar c1 s band shapes.13,27,28 these x - ray photoelectron spectrometry results are in accordance with the ultraviolet - visible analysis, revealing that the concentration of kmno4 directly influenced the degree of oxidation . We report a highly facile method for the preparation of scalable large - area go . The method was carried out at room temperature with the highest conversion possible (100% conversion of graphite flakes to go) to produce large - area go without ultrasonication . Here, we have identified key factors contributing to the large area of go formed, which are complete oxidation of graphite through a high degree of oxidation and the need to carry out the reaction at room temperature . Our method of synthesizing go will infinitely increase the rate of go production for numerous investigations, which might appeal to biologists, chemists, physicists, and materials scientists.
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In their landmark report, the institute of medicine noted that errors in health care are a significant cause of death and injury . In response to the national interest to reduce health care errors, the american college of obstetricians and gynecologists recommended the implementation of medication practices to improve patient safety . When implementing change for improvement, there is always the potential for adverse secondary effects, known as balancing measures . The hospital corporation of america (hca) has incorporated many patient safety initiatives including a conservative standardized oxytocin dosing regimen, part of which is a standard concentration of 15 units oxytocin per liter of intravenous fluid . Clark et al . Reported improved maternal and newborn outcomes with the implementation of the hca's oxytocin protocol . Subsequently, many hospitals including ours adopted a similar protocol in the interest of improving patient outcomes [6, 7]. In the past our labor and delivery unit had prepared oxytocin at a concentration of 20 units / l . With this concentration, the displayed rate on the infusion pump did not numerically match the dose of oxytocin that was being infused . For every 3.0 ml / hr of infusion, only 1.0 milliunit of oxytocin was infused per minute . Therefore our unit changed the oxytocin concentration as part of the hca's standardized oxytocin dosing protocol . The concentration of oxytocin was changed to 30 units of oxytocin per 500 ml of intravenous fluid . The concentration was standardized so that the infusion rate was numerically equivalent to the oxytocin delivery rate (i.e., 1.0 ml / hr = 1.0 milliunit / min). As a result, the standardized dosing related to postpartum oxytocin administration also changed . In the past a 1 liter infusion of normal saline with 40 units of oxytocin was given postpartum, and this was changed to a postpartum 500 ml infusion of normal saline containing 30 units of oxytocin . Shortly after the change, obstetricians began to verbally report a perceived increase in postpartum hemorrhage . This was attributed to the decrease postpartum oxytocin dose from 40 units to 30 units . The subjective impression of the obstetricians was that a postpartum dose of 30 units of oxytocin was insufficient and was causing an increase in the rate of postpartum hemorrhage . The purpose of this study was to evaluate a balancing measure of a quality improvement initiative to standardize the dosing of oxytocin; namely, whether there was an unanticipated consequence of an increase in postpartum hemorrhage . This is a retrospective, cohort study comparing the eight months preceding the change in medication protocol to eight months following the change in protocol . The study protocol was approved by the miami valley hospital institutional review board . As the study was of existing data and there was little or no risk for compromise of protected health care information, the requirement for written performed consent was waived . A search for the electronic medical record was performed in order to extract the pre and posthemoglobin values of all deliveries for the months of february 2009 until september 2009 pre and november 2009 through june 2010 post, omitting the month between these selected time frames to allow for education and adjustment to the protocol . In addition, all patients who had a vaginal or cesarean delivery and received blood transfusions during admission were identified . In the setting of a vaginal delivery, standard orders after the delivery of the placenta included a dose of 40 units of oxytocin in 1000 ml of lactated ringers (lr) prior to the change and 30 units of oxytocin in 500 ml of lr after the change . The oxytocin infusion was administered at 333 cc per hour for the duration of the third stage of labor and then reduced to 150 cc / hr for the remainder of the infusion . Women undergoing a cesarean delivery received the same amount of oxytocin (either 40 units in 1000 ml lr or 30 units in 500 ml lr) which was begun in the operating room at 333 cc / hr and then completed in the recovery room at 150 cc / hr . Total time for oxytocin for both vaginal and cesarean deliveries was 2 - 3 hours . Per anesthesia protocol, women undergoing a cesarean delivery received an additional 20 units of oxytocin in the operating room after delivery of the placenta . The practice of administering an extra 20 units of oxytocin in the operating room did not change between the two - time periods . Women with postpartum hemorrhage received additional doses of oxytocin and other uterotonic agents including methylergonovine, prostaglandin f2 alpha, and prostaglandin e1, per the obstetrician's discretion . Per preset admission orders on the electronic medical record (emr), all inpatient deliveries obtain a complete blood count (cbc) on admission and a cbc on postpartum day 1 (ppd 1). The time that elapsed between the cbc obtained on admission and the ppd 1 cbc varied from 12 to 24 hours . Women who received a blood transfusion or who lacked an admission or ppd 1 hemoglobin were excluded . Graphpad prism (graphpad software, san diego, ca) was used for statistical analyses . The mean change in hemoglobin was compared between the two groups by student's t - test . The mean change in hemoglobin was calculated for each month and a best fit multiple regression polynomial curve was constructed for each time series and compared by the extra sum of squares f test . Women who received blood products were excluded from the hemoglobin analyses . The rates of blood transfusion in the two groups were compared using fisher's exact test . There were 73/3564 (2.0%) women who received blood transfusions in the pregroup and 64/3295 (1.9%) women in the postgroup, p = 0.8 . There was incomplete hemoglobin data on 377 deliveries (10.6%) in the pregroup and 336 (10.2%) in the post group, leaving 3114 and 2895 deliveries, respectively, for the hemoglobin analyses . The cesarean delivery rate was 31.1% in the pregroup and 30.0 in the postgroup . The mean change standard deviation in pre to postdelivery hemoglobin was 1.53 0.03 g / dl for the pregroup versus 1.52 0.05 g / dl for the postgroup 2, p = 0.68 . The trend over both eight - month periods was best modeled with a third - degree polynomial, and the curves were not statistically distinguishable, p = 0.34 . There were 1003/3114 (32.2%) women in the pregroup with a hemoglobin decrease of at least 2 g / dl, compared to 918/2895 (31.7%) in the postgroup, p = 0.7 . There were 261/3114 (8.4%) women in the pregroup with a hemoglobin decrease of 3 g / dl or greater, compared to 252/2895 (8.7%), p = 0.7 . Figure 2 displays the number of women per month who had a change in hemoglobin exceeding 2 g / dl and 3 g / dl . The series were modeled by a third - degree polynomial, and there was not a significant difference between pre and post for a hemoglobin change of 2 g / dl (p = 0.44) nor 3 g / dl (p = 0.21). In this study we evaluated a standard postpartum 30-unit oxytocin dose versus a 40-unit dose and found there was no difference in the need for blood transfusion, mean hemoglobin change, and hemoglobin changes of 2 or 3 g / dl, demonstrating that the new medication protocol did not increase the incidence of postpartum blood loss nor number of blood transfusions despite administering a lower total dose of oxytocin . The effects of medical errors are far reaching and costly and have the potential for high liability, especially when they occur on the labor and delivery unit . However it should be emphasized that while standardization can have medical advantages, there may also be unanticipated disadvantages, that is, balancing measures . In the united states the prophylactic use of oxytocin during the third stage of labor for the prevention of uterine atony and postpartum hemorrhage is the accepted practice . Despite this wide spread practice there is insufficient data and little agreement or evidence to recommend an optimal dose of oxytocin and 10 to 40 units are usually given [10, 11]. Compared three different third - stage oxytocin doses (80 units, 40 units, or 10 units) and did not find a difference in the incidence of uterine atony or postpartum hemorrhage . However the 80-unit dose was found to reduce the need for treatment of hemorrhage after the first postpartum hour and fewer women had a decline of hematocrit of 6% or more . The strength of this study lies in its large sample size, as outcomes from approximately 6000 deliveries were included in analysis . In addition, the use of the electronic medical record allowed for accuracy in assessment of pre and postpartum hemoglobin levels as well as rates of transfusions . This study had several limitations which are attributable to the retrospective design: (1) the subjects were not stratified by mode of delivery; however, the percentage of cesarean deliveries during the two periods were not significantly different, (2) the baseline characteristics of the sample populations were not examined in detail, and (3) general assumptions are made regarding similarities between the cohorts studied . The two cohorts were not evaluated with respect to any differences regarding the existence of maternal bleeding disorders or other factor such as antepartum or intra - partum complications, which might increase the risk of postpartum hemorrhage . The study does not take into account changes in provider practice over time, repeated administration of oxytocin in the postpartum period, nor the use of additional uterotonics . The use of admission and postpartum day 1 hemoglobin values also has some limitations as it is not a direct measurement of blood loss, but its use has been reported in the literature as a surrogate to measuring actual blood loss [1214]. We have demonstrated that standardized oxytocin dosing resulting in a total decreased amount of medication delivered in the postpartum period does not result in an increased amount of postpartum hemorrhage . While this study exemplifies one aspect of obstetric care that has not been affected by medication standardization, many more avenues for research exist . Certainly, as quality improvement moves to the forefront of hospital evaluation and performance measurements, one can expect to see more and more implementations meant to simplify procedure and minimize error . It is imperative that we remain cognizant of the potential unanticipated adverse effects that may occur with new protocols.
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The proportion of adults over the age of 65 is expected to increase over the next 40 years . An anticipated rise in the number of older adults is expected to lead to an increase in the prevalence of age - related diseases, which in turn, might result in escalating health care costs and heightened distress among family and caregivers . Cognitive impairment, and more specifically alzheimer's disease, is one of the most threatening age - related diseases, but even so - called normal age - related cognitive decline can cause agonizing distress and loss of personal identity . Unfortunately, pharmaceutical treatments or preventions for cognitive impairment are only modestly effective, resulting in the search for nonpharmaceutical approaches such as intellectually stimulating activities, dietary interventions, and physical activity, for preventing or treating cognitive decline . A recent report estimated that modifiable risk factors including education, smoking, mid - life obesity, hypertension, diabetes, depression, and physical inactivity contribute significantly to the risk of alzheimer's disease, and that a 10% to 25% reduction in these factors could prevent as many as 3 million cases worldwide . Yet, despite the recognition of the importance of modifiable risk factors in the incidence and prevalence of cognitive impairment, there is often a misunderstanding of the research that has been conducted examining whether intervening on these modifiable risk factors would have any noticeable effect on brain or cognitive health . In contrast, a good deal of research has been conducted to examine the effects of physical activity and cognitive stimulation on human brain morphology and function . The aim of this review is to summarize recent research findings that examine the potential for physical activity, cardiorespiratory fitness, and exercise interventions to enhance brain health in late life . The studies reviewed here support the position that physical activity influences the endogenous pharmacology of the brain and takes advantage of the brain's natural capacity for plasticity, well into late adulthood . Physical activity increases the lifespan, reduces the risk for many cardiovascular diseases and cancers, and also reduces the risk for cognitive decline and depression in late adulthood . In short, we argue that the influence of physical activity on brain plasticity might have consequences not only for memory and other cognitive functions, but also has implications for many different psychiatric and neurologic conditions through a set of common biological pathways . First, studies using rodent models for exploring the ways in which physical activity influences the brain can control when and how much physical activity the animal receives . Hence, the nature of these systematic experiments allows for causal and directional conclusions about the effects of physical activity on learning and memory, neurotransmitter systems, metabolic and growth factors, and cell proliferation . Second, animal models allow for an examination of the cellular and molecular events resulting from physical activity that are simply impossible to study in humans . For these reasons, it is important to describe this literature since it provides a causal and low - level biological foundation to understand the effects observed in human neuroimaging and clinical studies . One of the earliest studies found that animals that were provided access to a running wheel in their cage tended to outperform their more sedentary counterparts on several different learning and memory tasks such as the t - maze and morris water maze . In one version of the morris water maze, rodents are made to swim in an opaque pool until they find the location of a submerged platform that sits just below the surface . By using cues located around the room, the rodent learns to navigate to the submerged platform more quickly after successive trials . In this task, both older and younger animals engaging in exercise demonstrate faster learning of the location of the submerged platform compared with rodents not engaging in exercise . Importantly, performance on the morris water maze has been frequently linked to the hippocampus, a medial temporal lobe structure critical in memory formation . In fact, other studies utilizing hippocampus - sensitive tasks have also reported that exercise enhances both acquisition and rtention, suggesting that the hippocampus might be especially sensitive to the effects of exercise . There is now substantial support for robust and consistent effects of physical activity on the morphology and function of the hippocampus . For example, one of the most consistent findings in this literature is that exercise has the capacity to increase cell prolifration in the dentate gyrus of the hippocampus, even in aged animals . These studies indicate that the hippocampus remains highly modifiable throughout the lifespan and that exercise has the capacity to take advantage of the plasticity of this structure . Cell proliferation in the hippocampus leads to an increased demand for nutrients to support the new neural architecture . After exercise, increased vascularization has been routinely found in several different brain regions including the cerebellum, motor cortex, hippocampus, and frontal cortex . Increased proliferation of cells and capillaries in the hippocampus work in concert to enhance learning and memory in behavioral paradigms, but these effects can also be observed on the neurophysiological level . For example, exercise increases the number of synapses in the hippocampus, enhances indices of long - term memory formation, and elevates the rate of gene expression for molecules associated with learning and memory such as brain - derived neurotrophic factor (bdnf) and serotonin . It is clear from this literature that exercise influences the integrity of the hippocampus by influencing gene expression, cell proliferation and survival, vascularization, and synaptic plasticity . However, this literature has identified many different brain regions influenced by exercise, indicating that exercise has widespread effects . In conclusion, there are many different molecular and cellular pathways mediating the effects of exercise on cognitive and behavioral outcomes, including increased neurogenesis, angiogenesis, and the production of growth factors important in memory and cognitive function . Greater amounts of physical activity and higher cardiorespiratory fitness levels are associated with better cognitive function in older adults . For example, older adult athletes outperform their more sedentary peers on many different cognitive tasks, and fitter individuals are faster and more accurate on executive functioning and memory tasks . Longitudinal studies of physical activity have also found that engaging in a greater amount of physical activity earlier in life is associated with better cognitive function later in life, with larger effects for individuals engaging in more intense exercises . However, cross - sectional and longitudinal observational studies are often plagued by confounding factors that make it challenging to make causal claims about the link between physical activity and cognitive function . In other words, in the studies described above it is equally likely that individuals with better cognitive function choose to participate in more physical activities than individuals with poorer cognitive function . Along these same lines, physical activity might instead be a proxy for better health habits more generally rather than being specific to physical activity per se . Randomized controlled trials reduce or eliminate some of the limitations of cross - sectional and observational studies . These types of interventions in which older adults are randomized to either a moderate intensity physical activity group or to a non - active or less - active control group, routinely demonstrate that increasing physical activity for 3 to 6 months is effective at improving cognitive performance . For example, in one study, inactive older adults were randomized to either a moderate intensity physical activity group or to a stretching and toning control group for 6 months . Both groups came into the laboratory 3 days per week and the exercise group participated in moderate - intensity exercises for 30 to 45 minutes per day while the stretching group participated in stretching exercises for the same amount of time . Trained exercise physiologists monitored heart rates, intensity, and compliance in both groups for the duration of the exercise regimen . This study found that participation in moderate - intensity physical activity (eg, brisk walking) was effective at enhancing performance on tasks that measured executive functions, but was less effective at improving performance on tasks that measured other cognitive domains . In contrast, the stretching and toning group did not show significant improvements in performance over this same period . Meta - analyses of physical activity interventions have confirmed that the effects of exercise on cognitive function in late life are both general and specific . General in the sense that many different cognitive domains are improved after several months of exercise, but specific in the sense that executive functions are enhanced more than other cognitive functions . First, in terms of cognitive function, the effects of exercise appear to be widespread, but most strongly associated with executive domains . This suggests that brain regions and networks that support executive functions might be more sensitive to the effects of exercise than other brain areas . The rodent literature largely supports this claim, with the largest and most consistent effects of exercise appearing in regions that support higher - level cognitive functions including the hippocampus, frontal cortex, and basal ganglia . The second key principle emerging from these studies is that the brain remains modifiable well into late adulthood, and physical activity has the capacity to take advantage of brain plasticity . Brain plasticity resulting from exercise can be detected at the molecular and cellular level in rodents and at the cognitive level in humans . These two points, specificity and plasticity, provide the foundation for neuroimaging methods to examine whether physical activity, fitness, or exercise has any appreciable effect on the morphology or function of the human brain . Given the principles described above, neuroimaging studies exploring these associations have hypothesized that physical activity would influence the morphology and function of the human brain and that the effects would be widespread but most consistently associated with regions that support higher - level cognitive functions such as the prefrontal cortex and hippocampus . One of the unfortunate characteristics of the brain is that it generally shrinks and atrophies with advancing age . In fact, both the prefrontal cortex and hippocampus shrink at roughly 1% to 2% annually in individuals over the age of 55, with more precipitous rates of atrophy when individuals begin experiencing cognitive impairment . Although the rate and trajectory of decline varies from region to region, the general finding is that regions that support memory and executive functions show the earliest and most rapid decline . Interestingly, the loss of brain volume is mirrored by age - related changes in cognitive function with the most significant losses occurring on memory and executive tasks . Yet, it is these cognitive domains and brain areas that appear the most sensitive to physical activity training . Would greater amounts of physical activity or higher cardiorespiratory fitness levels have any beneficial or positive associations with the morphology of the older adult brain? There have now been several studies finding that older adults who are more fit, more physically active, and who participate in exercise interventions have greater brain volumes than their less fit and less active counterparts . In one cross - sectional study, cardiorespiratory fitness levels were assessed in a sample of cognitively healthy older adults and voxel - based morphometry was used to assess gray matter volume . Although increased age was associated with reductions in gray matter volume throughout the prefrontal, temporal, and parietal cortices, these same brain regions showed less atrophy in adults that were more fit . These results demonstrated that remaining more aerobically fit could help to preserve brain tissue that would normally atrophy with age . Higher fitness levels have now been associated with greater gray matter volume in other populations, including postmenopausal women receiving hormone therapy, a higher educated older adult sample, a sample with multiple sclerosis, and older adults with mild cognitive impairment . These studies have all derived a similar conclusion from these results: individuals with a higher level of fitness have greater gray matter volume than less fit individuals, and the associations are relatively specific to brain areas that support higher - level cognition, including the prefrontal cortex . As described above, much of the animal literature has focused on the effects of exercise on hippocampal plasticity and memory functions supported by the hippocampus . This question is important since the hippocampus shrinks with advancing age and contributes to agerelated memory loss . In 165 cognitively normal older adults, cardiorespiratory fitness levels were recorded in addition to high - resolution anatomical images of the brain . The size of the hippocampus was assessed using an automated segmentation algorithm that uses a point distribution model to determine the location, size, and shape of the structure . A clear association was found between higher fitness levels and greater hippocampal volume, but importantly, greater hippocampal volume also mediated the fitness - memory association . This result suggests that greater hippocampal volume is not just a meaningless by - product of more vascularization, but rather has a meaningful impact on memory function in late life . This general association between higher fitness levels and larger hippocampal volume has now been replicated in individuals with mild cognitive impairment . Cross - sectional research defines important associations between variables of interest, such as cardiorespiratory fitness levels and cortical volume . Demonstrating these associations is necessary before embarking on a lengthy and expensive longitudinal randomized trial . However, there are inherent limitations to cross - sectional designs that prohibit the ability to draw conclusions about the causal nature of physical activity on brain plasticity . Several studies for example, in the cardiovascular health study at the pittsburgh, pennsylvania site, 1479 ambulatory adults over the age of 65 were enrolled into a longitudinal study on the incidence of cardiovascular diseases . Information about lifestyles and physical function were collected as part of this study including information on the frequency and duration of walking . Approximately 9 years after the original enrollment period these same participants were recruited to participate in a brain mri study in which high - resolution brain images were collected . The brain images from 299 cognitively normal adults were selected from this sample and used in an analysis to examine whether greater amounts of self - reported walking 9 years earlier was predictive of gray matter volume later in life . The analysis of this data confirmed that greater amounts of physical activity was associated with greater gray matter volume in several different brain regions including the frontal cortex, parietal cortex, and temporal cortex including the hippocampus . Interestingly, after a period of 4 more years, 116 of these 299 adults were diagnosed with either mild cognitive impairment or dementia, but greater graymatter volume associated with physical activity was associated with a two - fold reduced risk of developing cognitive impairment . This study demonstrated for the first time the link between participation in physical activityearlier in life, greater gray matter volume, and the reduced risk for cognitive impairment later in life . This study and others demonstrate that the effects of physical activity on brain plasticity might endure and influence the risk for cognitive impairment over a span of several years . Randomized interventions have also reported that assigning sedentary older adults to engage in more physical activity results in an increase in graymatter volume in several different brain areas . For example, colcombe et al randomized a group of cognitively normal adults to either a moderate - intensity walking exercise program or to a stretching and toning control group . Similar to the study described above, this study required participants to report to the laboratory three times per week for a period of 6 months . High - resolution brain mri scans were collected both before and after the intervention period . Interestingly, the walking exercise group showed a significant increase in the volume of prefrontal and temporal brain areas along with an increase in the volume of the frontal white matter tracts especially the genu of the corpus callosum . Another randomized intervention of physical activity examined whether participation in 1 year of a structured exercise regimen would increase the volume of the hippocampus in older adults . In this study, 120 cognitively normal older adults participated in a similar exercise design as that described previously . High - resolution brain scans were collected before the intervention, after 6 months, and then at completion of the 1-year trial . Although the thalamus and caudate nucleus did not show significant changes in volume resulting from exercise, there was an effect of exercise on the size of the hippocampus . Whereas the stretching and toning control group displayed about a 1.4% decline in the size of the hippocampus the exercising group showed an increase of about 2% over this same 1-year period . This study demonstrated that the volume of the hippocampus remains modifiable into late adulthood, and participation in 1 year of consistent and moderate intensity exercise was sufficient for increasing the size of the structure . Furthermore, the changes in hippocampal volume for the exercising group were correlated with improvements in memory performance suggesting an important link between changes in volume induced by exercise and memory enhancement . In fact, more recent studies have found that the volumetric differences observed as a function of cardiorespiratory fitness mediate improvements in memory and executive function, again supporting the claim that these volumetric effects are not meaningless by - products, but important factors in promoting better cognitive function . This discussion summarizes the relatively well - established scientific literature using cross - sectional, longitudinal, observational, and randomized controlled trials examining the effect of physical activity or cardiorespiratory fitness on regional gray matter volume . These studies have consistently reported that higher fitness levels are associated with larger brain volumes, and that participation in only modest amounts of physical activity is sufficient for increasing gray matter volume in select brain regions . In addition, these results are in line with the animal literature and human cognitive literature described in preceding sections demonstrating the brain plasticity and specificity of the effects of greater amounts of physical activity . Volumetric data has proven useful in identifying how physical activity could alter the morphology of the adult brain . However, other neuroimaging methods including functional magnetic resonance imaging (fmri) and resting state connectivity (rs) mri approaches allow for an investigation of the effects of physical activity on brain network dynamics . In one of the earliest studies to examine this, colcombe et al employed a task measuring selective attention and executive control in a two - part experiment . In the first experiment, higher cardiorespiratory fitness levels were associated with better performance on the task and this was paralleled by increases in fmri activity in the dorsolateral prefrontal and parietal brain regions . The second experiment was a randomized exercise intervention in which adults were assigned to either receive a structured exercise regimen for 6 months or to a stretching and toning control group for the same amount of time . The participants performed the same selective attention task as the participants in the first experiment . The results from the randomized trial were strikingly similar to the results from the crosssectional study . That is, after 6 months of the intervention, the exercise group showed increased activity in the dorsolateral prefrontal cortex and parietal cortex and decreased activity in areas that support conflict monitoring such as the anterior cingulate cortex . These results are important because they demonstrate that in addition to volumetric changes resulting from exercise there are also significant changes in task - evoked brain function . Hence, the brain processes task demands more efficiently after only 6 months of exercise . Although there are only several published studies using fmri paradigms, each of these studies has found increased fmri activity in prefrontal regions including during a semantic memory task, the digit symbol substitution task, and the stroop task as a function of either higher cardiorespiratory fitness levels or greater physical activity levels . Yet, each of these studies also recognizes the complex nature of cognitive function and the necessity of understanding the networks of regions that support cognition and how physical activity exerts its effects on these networks . Hence, it is important to identify not only which brain areas are associated with physical activity, but also to understand how the communication between regions is influenced by physical activity . Could the functional connectedness of the network improve after several months of exercise and would these effects mediate improvements in memory and executive function? Regions of interest can be used as seeds to examine whether regions that are functionally connected with the seed region vary as a function of some variable of interest (eg, cardiorespiratory fitness levels). Using a seed - based approach to examine functional connectivity, voss et al found that older adults that had higher cardiorespiratory fitness levels had greater connectivity in the so - called default mode network . Further, they found that increased connectivity mediated the fitness related enhancements of executive control . Since the default mode network is reduced in older adults with mild cognitive impairment and dementia, increased functional connectivity indicates that physical activity might reduce the risk of impairment by elevating the cohesiveness of the default mode network . In fact, results from two randomized interventions indicate that the functional connectivity of these networks can be modified after several months of physical activity . The studies described above focus on three forms of brain health and integrity in late life: morphology, task - evoked functional dynamics, and connectivity . For each of these measures, cross - sectional, observational, and randomized interventions indicate that physical activity is capable of modifying age - related losses and that physical activity - induced changes in brain integrity and function mediate improvements in cognition . In summary, the human neuroimaging literature on physical activity indicates that the brain remains modifiable into late adulthood, the effects are distributed throughout the brain, but are most robust in the prefrontal and medial temporal lobe regions . The cochrane collaboration performed a systematic review of the effects of exercise on depression in adults of mixed ages . They identified 32 trials (1858 participants) that fulfilled their inclusion criteria, of which 30 (1101 participants) provided data for meta - analyses . Based on these 30 trials, the authors concluded that exercise seems to improve depressive symptoms in individuals with depression when compared with no treatment or a control intervention . In comparison to no treatment, exercise had a moderate effect size (standardized mean difference [smd] -0.67, 95% confidence (ci) -0.90 to -0.43). There were no significant differences when comparing the effects of exercise with cognitive therapy or antidepressant treatment . When the authors limited the analyses to the four high - quality trials (326 participants), the pooled smd was -0.31 (95% ci -0.63 to 0.01) indicating a small effect in favor of exercise . Among the 32 trials identified that fulfilled the inclusion criteria, 8 studies were focused on or included adults older than 60 years six of the studies involved aerobic exercise and two studies progressive resistance training . Of the 6 studies that involved aerobic exercise, various exercise and comparator interventions blumenthal and colleagues (1999) studied community volunteers with major depressive disorder (mdd) (n=156) mean (sd) age of 57 (6.5) randomized to aerobic exercise (group walking or jogging 3 times per week), antidepressant pharmacologic treatment (sertraline), or the combination . They found that all treatment groups had statistically significant improvement in depression scores, although participants receiving medication alone had the fastest initial response . After 16 weeks of treatment, exercise was equally effective in reducing depression among older adults with mdd . A limitation to this study was the absence of a placebo or control intervention . In a follow - up study, blumenthal and colleagues examined community - dwelling older adults with mdd (n=202), mean (sd) age 52 (8), randomly assigned to home - based exercise, supervised exercise in a group setting, sertraline, or placebo for 16 weeks . While there was a high placebo response rate, the efficacy of exercise was comparable to antidepressant pharmacotherapy, and both were better than placebo . Brenes and colleagues studied 37 older adults with a mean (sd) age of 73.5 (7.8) with minor depression, randomized to exercise, antidepressant pharmacotherapy (sertraline), or usual care over 16 weeks . In the 32 participants who completed the study, they found trends for exercise and sertraline to be superior to usual care in improving emotional and physical functioning . Mather and colleagues examined whether exercise is effective as an adjunct to antidepressant pharmacotherapy in older adults . Eighty - six older adults with depression (mean age 65) were randomly assigned to attend exercise classes or health education talks for 10 weeks . At 10 weeks, a significantly higher proportion of the exercise group (55% versus 33%) experienced a greater than 30% decline in depressive symptoms as measured with the hamilton rating scale for depression . Mcneil and colleagues (1991) randomly assigned 30 community dwelling, moderately depressed older adults with a mean (sd) age of 72.5 (6.9) to 1 of 3 interventions: experimenter - accompanied exercise (walking), social contact control condition, and a wait - list control . They found that exercise and social contact both resulted in reductions in the beck depression inventory . Lastly, williams and tappen examined the effects of exercise training for depressed older adults with alzheimer's disease . Subjects were randomly assigned to 16 weeks of comprehensive exercise, supervised walking, or social conversation . They found that all three groups had a reduction in depressive symptoms, with exercise showing a slightly greater benefit . Two studies found antidepressant effects of progressive resistance training in older adults with depression . Singh and colleagues studied the effects of progressive resistance training (prt) on depressed adults 60 years and older with a mean (sd) age of 71.3 (1.2). Over 10 weeks compared with an attention - control group, prt was associated with an improvement in the measures of depressive symptoms, quality of life, social functioning, and strength . In a follow - up study, singh and colleagues found that higher - intensity prt was more effective than low - intensity prt on depression in older adults . These studies support the argument that exercise has antidepressant effects in older adults, yet the mechanism of action remains unclear . The studies are careful to note the potential for the antidepressant effects of expectation and attention in research participation as well as for socialization when engaging in group exercises . Further, investigators have also suggested the effects of increased self - efficacy, a sense of mastery, positive thoughts, distraction from negative thoughts, and enhanced self - concept . However, biological mechanisms related to overall brain health are also likely related to the mood elevating properties of exercise . These mechanisms, described earlier, include enhanced gray matter volume in prefrontal cortex and hippocampal brain areas, elevated functioning of brain circuits involved in mood and emotional function such as subregions of the frontal cortex and medial temporal lobe, and improvements in functional connectivity of the default - mode network . In addition, it is also likely that exercise is having a pleiotropic effect on molecular systems related to the hypothalamic - pituitary - adrenocortical axis, dopaminergic, noradrenergic, serotonergic neurotransmission, immune function, and bdnf (figure 1). However, these biological mechanisms of exercise have not yet been carefully studied in older adults with depression . The role that changes in morphology and function could have on mitigating depressive symptoms remains speculative at this time . In this review we have briefly summarized the expansive and ever - growing literature on the effects of physical activity on brain health and plasticity . We can conclude from this overview that physical activity has consistent and robust effects on the brain, which mediate improvements in cognitive performance and reduce the risk for neuropsychiatric disorders . The beauty of this research is that the effects appear consistent across species and populations indicating an exceptional level of translation that is rare to find in other disciplines . The research in this area has demonstrated that the effects of physical activity on brain plasticity in late life has a remarkable degree of specificity, such that some brain areas appear to be more commonly or easily influenced by physical activity than other areas one explanation might be that the hippocampus, frontal cortex, and neighboring areas are more inherently plastic than other brain areas and that the degree of specificity is simply a characteristic of the brain regions examined rather than anything specific to the capabilities of physical activity per se . However, an alternative explanation is that these brain areas contain some molecular or cellular process that is influenced by participation in physical activity . For example, bdnf has been described as one possible molecular pathway by which exercise improves cognitive function, but bdnf levels are found in different concentrations throughout the brain with higher levels in the hippocampus and cerebellum than in other areas . A third possible explanation could be that the brain areas that show the most amount of atrophy with age, including the frontal cortex and hippocampus, are the most sensitive to the effects of physical activity . Thus, the specificity of physical activity on the frontal cortex and hippocampus might be related to the atrophying nature of these areas . According to this reasoning, since these brain areas are shrinking with age, there is more room for them to grow with an intervention like physical activity . Therefore, the specificity of physical activity has to do with relatively little variation in the size and function of other brain regions with advancing age . Whatever the explanation for specificity, the effects appear to be both robust and consistent across samples and populations . This indicates that these effects are unlikely to be confounded by a particular sample characteristic (eg, gender) or comorbidity (eg, depression) and more likely reflects an adaptive biological importance of physical activity to enhance and maintain the body's organs, including the brain . Despite the well - established literature linking physical activity to brain health and plasticity in late life, first, although a number of studies described above have found effects with moderate intensity exercise for several months, the exact dose - response nature of the link between physical activity and mood, cognition, or brain health remains unknown . In other words, there is a very poor understanding of how much physical activity is necessary to observe effects . Second, individuals stop exercising for a variety of different reasons including injuries, illness, and personal issues (eg, mourning). Because of this it is important to examine whether the effects of a physically active lifestyle are retained or lost after some period of inactivity . Unfortunately we have a very poor understanding of the retention of the effects of physical activity . Third, we have a very poor understanding of the types of exercises that might be most useful to promote a healthier brain . It is conceivable that competitive sports like tennis offer additional benefits beyond noncompetitive sports because of their dependence on physical coordination, cognitive effort, and social interaction . In sum, although we have a solid understanding of the potential for physical activity to enhance cognitive and brain health in late life there remain many unanswered questions for future research to pursue.
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Cutaneous leishmaniasis (cl) is an endemic parasitic disease in middle - east countries including iran . Most skin lesions evolve from papular, nodular, and plaques . However, there are rare clinical forms including, disseminated cl (dcl), sporotrichoid, and lupoid which are related to host immunity . Delayed - type hypersensitivity is an important feature of the disease pathogenesis, and it can be measured by leishmanin skin test (lst), also called montenegro test . In disseminated type, lst and other tests of specific cellular immunity are negative, and affected patients with lupoid leishmaniasis have a vigorous cellular immune response but low antibody titer . In sporotrichoid type of cl, there is lymphatic dissemination in the form of subcutaneous nodules (scn). However, there is no report of evaluation of host cellular immunity in these cases . The aim of this study was to determine and compare the patients reactivity to lst in three forms of cl, including common plaque type [figure 1], lupoid type [figure 2], and sporotrichoid type [figure 3]. This was a descriptive cross - sectional study performed in skin disease and leishmaniasis research centre, isfahan, iran . The patients enrolled in this study had cl confirmed by positive direct smear of their lesions and had three clinical forms of common plaque type, lupoid type, and sporotrichoid type . Patients more than 60 and less than 5 years old, those with any kind of state which affects the immune system such as underlying disease, pregnancy, hypersensitivity disorders, and steroids intake and clinical course shorter than 2 months were excluded from the study . After being informed about the study, the patients or the guardians signed a consent form, and then lst was performed for them . Each test vial contains 1 ml phosphate buffered saline, 0.1% thiomersal, and 610 6 killed l.major promastigotes . Lst was carried out by injecting 0.1 ml of a suspension of antigen intradermal with a 27-g needle in volar aspect of right forearm . In order to minimize the placebo effect, we also injected a control solution supplied from the same institute containing 1 ml pbs and 0.1% thiomersal in the contralateral side . The reaction was read after 48 h and the induration was measured by ballpoint technique . In the case of no reactivity, the test would be observed at 72 h of the injection . Results were categorized as negative (0 - 5 mm induration), positive (6 - 14 mm), and strongly positive (15 mm). With a special notice to the fact that in the site of injection of control solution, there should be no reaction . (chicago, usa) version 16 software using kruskal wallis and analysis of variance . Totally, 500 patients suspected to cl were referred to skin diseases and leishmaniasis research center from august 2010 to december 2011, of which 200 parasitologically confirmed cases with three different types of cl were enrolled in the study and underwent lst . Hundred and forty - three patients had common plaque type, 24 patients had lupoid type, and 33 were diagnosed with sporotrichoid type of cl . The mean duration of the disease was 5.99 1.04 (min: 2 months and max: 96 months). In the group with plaque type, 86% had a positive lst, 13.3% were negative, and 0.7% were strongly positive . In the lupoid group, these figures were 45.8%, 8.4%, 45.8%, respectively . In the sporotrichoid group, lst was positive in 27.3%, negative in 72.7%, and none of the patients had a strongly positive reaction (p <0.05). The minimum mean induration was in sporotrichoid group which was 3.30 2.24 mm and the maximum mean induration with 11.2 6.10 mm belonged to the patients with lupoid type [table 1; figures 4 and 5]. The results of leishmanin skin test in three groups of cutaneous leishmaniasis with different clinical forms positive leishmanin skin test exaggerated positive leishmanin test in lupoid type the lst or montenegro test is an important tool for the diagnosis and epidemiological surveys of cl . Because of the high sensitivity of lst in cl the test becomes positive a few weeks after infection and it is a decisive method for the diagnosis of older leishmanial lesions, when the number of parasites is low and therefore, difficult to detect . The test is useful for follow - up in vaccination programs, and is also used as a parameter for evaluating the development of immune protection . The lst consists of the intradermal injection of a suspension of antigen prepared from dead promastigotes, and relies on the cell - mediated immune response that usually occurs with cl . The reaction read at 48 - 72 h. understanding the timing of lst is of significant importance to avoid false - negative results in those few cases that present with early disease; therefore, we excluded the patients with a clinical course shorter than 2 months . We also excluded patients with any kind of possible impairment in the immune system in order to avoid possible negative effects on test results . Protective immunity against leishmaniasis is largely, if not wholly t - cell mediated and associated with production of interferon gamma . In this study, the test was supplied from pastor institution, tehran, iran, where the safety and efficacy of it have been previously documented . It is negative in patients with very recent cl infection and dcl . Whereas in lupoid leishmaniasis which is a rare peculiar form of chronic cl, a vigorous cellular immunity response and low number of parasites in lesions have been shown . This recurrent form of the disease refers to the development of the new lesions within the scar of the healed lesions mimicking lupus vulgaris . Sporotrichoid type of cl is a clinical form of cl that represents lymphatic dissemination, a phenomenon not widely recognized in the form of scn and is seen in 10% of cl patients . The scn were usually inconspicuous, painless, proximal to the primary skin lesions when multiplied they show sporotrichoid configuration . The results of our study showed that most of the positive lst results belong to plaque and lupoid groups, the majority of strongly positives belong to lupoid, and the most of negative lst results were related to sporotrichoid type, the negativity of leishmanin test in some patients with plaque and lupoid types can be explained by the sensitivity of the test used which is 88% according to our results . In addition, the mean induration in sporotrichoid group was less than the other two groups . According to results obtained from this study it can be suggested that lupoid and sporotrichoid types of cl are parts of a continuous spectrum of the disease with an enhanced cellular immunity in lupoid form and a decreased state in sporotrichoid type.
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Osteoarthritis is a multifactorial disease, often caused by injury or repetitive trauma, and involves metabolic as well as inflammatory components . Constant wear - and - tear on joints leads to the release of phospholipids from damaged cells which are then converted by pla2 into aa . Dietary habits, particularly excess consumption of aa in the form of omega-6 fatty acids, have been shown to impact the extent and progression of oa in humans . Through the action of the cox and 5-lox enzymes, aa from tissue destruction and diet is converted into inflammatory metabolites such as thromboxane (tx), prostaglandins (pgs), prostacyclins (pcs), and leukotrienes (lts) [3, 4]. Flavocoxid, marketed as limbrel, is a usfda - regulated prescription medical food (cfr volume 21 usc [code] section 360ee (b)(3)) for the clinical dietary management of the metabolic processes of oa . The molecules in flavocoxid were isolated by high - throughput screening of thousands of natural extracts having anti - po, cox-1 and cox-2 activity as well as 5-lox enzyme inhibition . The product is composed of a proprietary mixture of the flavonoid molecules baicalin, extracted from scutellaria baicalensis, and catechin, from acacia catechu, concentrated to greater than 90% purity (figure 1). Recent clinical efficacy trials utilizing garcinia kola, pine bark extract - derived bioflavonoids [8, 9], and flavocoxid itself [1012] demonstrate a renewed interest in the use of natural molecules for the management of oa . Using a variety of in vitro enzyme, cell, gene expression, and antioxidant assays, we examined the anti - inflammatory mechanism of action of flavocoxid . Flavocoxid's inhibitory activity on pla2 as well as its exact interaction with the cox and 5-lox enzymes was clarified . In addition, the broad antioxidant capacity of flavocoxid and its effect on nitrite production in a cell model was determined and related to its damping of cox-2 gene expression compared to other anti - inflammatory agents . These pleiotropic, anti - inflammatory effects of flavocoxid are discussed, correlating this in vitro characterization to in vivo safety and efficacy results in humans for the clinical management of oa . The following nsaids were purchased from sigma - aldrich (st . Louis, mo, usa): meloxicam, sodium salt (#3935); naproxen, sodium salt (#5160); diclofenac, sodium salt (#d6899); ibuprofen, sodium salt (#i1892) and ns-398 (#n194). (whitehouse station, nj, usa), and valdecoxib and celecoxib from pfizer (new york, ny, usa). Flavocoxid, as well as 90% pure baicalin and 90% pure catechin, were provided by primus pharmaceuticals, inc . Each compound was dissolved in 100% dimethyl sulfoxide (dmso; sigma - aldrich). Therefore, all concentrations are given in g / ml for the compounds tested rather than micromolar concentrations . All procedures complied with the standards for care and use of animal subjects as stated in the guide for the care and use of laboratory animals (institute of laboratory animal resources, national academy of sciences, bethesda, md, usa). Peritoneal macrophages were obtained from male sprague dawley rats (250275 g) by washing the abdominal cavity with rpmi 1640 . The cells were centrifuged twice and resuspended in the same medium at a concentration of 3 10/ml macrophages were purified after 2 h adhesion to plastic petri dishes (nunc, roskilde, denmark) at 37c . The homogeneity and the viability of macrophages were greater than 98% as determined by differential staining and trypan blue exclusion . Macrophages were stimulated for 6 h with 1 g / ml of lipopolysaccharide (lps). Lipopolysaccharide - stimulated macrophages were coincubated with flavocoxid at 10, 20, 50, 100, 200, and 500 g / ml or rpmi medium alone . Cell viabilitycell viability of peritoneal macrophages, following exposure to flavocoxid (from 10, 20, 50, 100, 200 and 500 g / ml) and/or with 1 g / ml lps at 37c, was determined after 24 h of incubation by the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, as described by mosmann . Cell viability of peritoneal macrophages, following exposure to flavocoxid (from 10, 20, 50, 100, 200 and 500 g / ml) and/or with 1 g / ml lps at 37c, was determined after 24 h of incubation by the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, as described by mosmann . Phospholipase a2 assayphospholipase a2 was assayed in cytosolic extract by using the cpla2 assay kit (cayman, ann arbor, mich, usa) following the manufacturer's protocol . Briefly cells were homogenized in lysis buffer (50 mm hepes ph 7.4, 1 mm edta), sonicated and then centrifuged at 10.000 g for 15 min at 4c . Absorbance was read at od414 nm, and results were compared to the positive control (bee venom pla2). Phospholipase a2 was assayed in cytosolic extract by using the cpla2 assay kit (cayman, ann arbor, mich, usa) following the manufacturer's protocol . Briefly cells were homogenized in lysis buffer (50 mm hepes ph 7.4, 1 mm edta), sonicated and then centrifuged at 10.000 g for 15 min at 4c . Absorbance was read at od414 nm, and results were compared to the positive control (bee venom pla2). Cox-1 and cox-2 cyclooxygenase inhibition assayflavocoxid was dissolved in 100% dmso at a stock concentration of 10 mg / ml . Flavocoxid was tested in triplicate at 0, 0.1, 1, 10 and 50 g / ml . Dmso alone alters oxygenase activity . Therefore, the highest concentration of flavocoxid that could be tested using the oxygraph assay (cayman chemical company, inc .) To determine co - specific inhbition without interference was 50 g / ml . Indomethacin (mw = 357.8) and ns-398 (mw = 314.4) were run as positive controls for inhibition of cox-1 and cox-2, respectively . Indomethacin was tested in triplicate at 0, 3.6, 18, 36, 180, and 360 ng / ml . Ns-398 was tested in triplicate at 0, 3.1, 15.7, 31.4, 157 and 314 ng / ml . These inhibitors were pre - incubated with the enzyme in reaction buffer for one minute prior to the addition of aa . Assays were performed using 100 units of ovine cox-1 or ovine cox-2 (one unit of enzyme consumes one nanomole of oxygen per minute at 37c in 0.1 m tris - hcl buffer, ph 8.0, containing 20 m aa, 5 mm edta, 2 mm phenol, and 1 m hematin). Assays were initiated by the addition of 20 m aa, and oxygen consumption measured using a gilson model 5/6h oxygraph equipped with a clark oxygen electrode . Flavocoxid was dissolved in 100% dmso at a stock concentration of 10 mg / ml . Flavocoxid was tested in triplicate at 0, 0.1, 1, 10 and 50 g / ml . Dmso alone alters oxygenase activity . Therefore, the highest concentration of flavocoxid that could be tested using the oxygraph assay (cayman chemical company, inc .) To determine co - specific inhbition without interference was 50 g / ml . Indomethacin (mw = 357.8) and ns-398 (mw = 314.4) were run as positive controls for inhibition of cox-1 and cox-2, respectively . Indomethacin was tested in triplicate at 0, 3.6, 18, 36, 180, and 360 ng / ml . Ns-398 was tested in triplicate at 0, 3.1, 15.7, 31.4, 157 and 314 ng / ml . These inhibitors were pre - incubated with the enzyme in reaction buffer for one minute prior to the addition of aa . Assays were performed using 100 units of ovine cox-1 or ovine cox-2 (one unit of enzyme consumes one nanomole of oxygen per minute at 37c in 0.1 m tris - hcl buffer, ph 8.0, containing 20 m aa, 5 mm edta, 2 mm phenol, and 1 m hematin). Assays were initiated by the addition of 20 m aa, and oxygen consumption measured using a gilson model 5/6h oxygraph equipped with a clark oxygen electrode . Cox-1 and cox-2 peroxidase inhibition assaysa cleavable, peroxide chromophore (n, n, nn-tetramethyl - p - phenylenediamine dihydrochloride (tmpd)) (sigma) was included in the assay to measure the inhibitor effect on po activity of cox-1 and cox-2 in the presence of aa as a cofactor . Each inhibitor was tested for po - specific inhibition in triplicate (cambridge biomedical research group, inc . ), at room temperature (25c) over a range of concentrations from 0 to 500 g / ml using ovine cox-1 and cox-2 enzymes (provided by cayman chemical, ann arbor, mich, usa), hematin (sigma), tmpd (sigma), and aa (sigma). The mean results for each inhibitor concentration versus percentage inhibition was plotted, and the ic50 determined by taking the half - maximal point along the isotherm and intersecting the concentration on the x - axis . A cleavable, peroxide chromophore (n, n, nn-tetramethyl - p - phenylenediamine dihydrochloride (tmpd)) (sigma) was included in the assay to measure the inhibitor effect on po activity of cox-1 and cox-2 in the presence of aa as a cofactor . Each inhibitor was tested for po - specific inhibition in triplicate (cambridge biomedical research group, inc . ), at room temperature (25c) over a range of concentrations from 0 to 500 g / ml using ovine cox-1 and cox-2 enzymes (provided by cayman chemical, ann arbor, mich, usa), hematin (sigma), tmpd (sigma), and aa (sigma). The mean results for each inhibitor concentration versus percentage inhibition was plotted, and the ic50 determined by taking the half - maximal point along the isotherm and intersecting the concentration on the x - axis . 5-lox inhibition assaya lipoxygenase inhibitor assay kit (cayman chemical) was used in which potato 5-lox (cayman chemical) was substituted for the soy 15-lox usually present in the kit . The buffer was also changed from phosphate buffer, ph 6.8 to a tris - hcl, ph 7.4 buffer and was used to measure 5-lox inhibition for each inhibitor . This assay detects the formation of unstable hyperoxides, hpetes, which are then converted to lts through an oxygen sensing chromagen system consisting of feso47h2o and nh4scn that changes to bright yellow . The assay was performed in triplicate using potato 5-lox (15.3 units), linoleic acid (la) as a cofactor, and tmpd as a substrate with a variety of inhibitors titrated from 0 to 500 g / ml and flavocoxid from 0 to 1000 g / ml at room temperature . The mean results for each inhibitor concentration versus percentage inhibition was plotted and the ic50 determined by taking the half - maximal point along the isotherm and intersecting the concentration on the x - axis . A lipoxygenase inhibitor assay kit (cayman chemical) was used in which potato 5-lox (cayman chemical) was substituted for the soy 15-lox usually present in the kit . The buffer was also changed from phosphate buffer, ph 6.8 to a tris - hcl, ph 7.4 buffer and was used to measure 5-lox inhibition for each inhibitor . This assay detects the formation of unstable hyperoxides, hpetes, which are then converted to lts through an oxygen sensing chromagen system consisting of feso47h2o and nh4scn that changes to bright yellow . The assay was performed in triplicate using potato 5-lox (15.3 units), linoleic acid (la) as a cofactor, and tmpd as a substrate with a variety of inhibitors titrated from 0 to 500 g / ml and flavocoxid from 0 to 1000 g / ml at room temperature . The mean results for each inhibitor concentration versus percentage inhibition was plotted and the ic50 determined by taking the half - maximal point along the isotherm and intersecting the concentration on the x - axis . Nitrite productionnitrite levels, a breakdown product of nitric oxide (no), were measured in a standard griess reaction . Briefly, 100 l of lps - stimulated macrophage supernatants with and without flavocoxid were incubated with an equal volume of griess reagent (1% sulphanilamide and 0.1% naphthyl - ethylenediamine dihydrochloride in 2.5% phosphoric acid). After 10 min of incubation at room temperature, the absorbance of the chromophore was measured at od540 nm using a microtitre plate reader . Nitrite concentrations were calculated by comparison with a standard calibration curve with sodium nitrite (nano2: 1.26 to 100 mm), with control baseline supernatant as the blank . Nitrite levels, a breakdown product of nitric oxide (no), were measured in a standard griess reaction . Briefly, 100 l of lps - stimulated macrophage supernatants with and without flavocoxid were incubated with an equal volume of griess reagent (1% sulphanilamide and 0.1% naphthyl - ethylenediamine dihydrochloride in 2.5% phosphoric acid). After 10 min of incubation at room temperature, the absorbance of the chromophore was measured at od540 nm using a microtitre plate reader . Nitrite concentrations were calculated by comparison with a standard calibration curve with sodium nitrite (nano2: 1.26 to 100 mm), with control baseline supernatant as the blank . Antioxidant assaysthe in vitro antioxidant activity of flavocoxid was evaluated using oxygen radical absorbance capacity (orac) procedures (brunswick laboratories, norton, mass, usa). Values are expressed as mol of trolox equivalents (te) per g of dry weight and compared to known values for vitamins c and e. the orac analysis provides a measure of the scavenging capacity of antioxidants against the peroxyl radical, which is one of the most common ros found in the body . Orachydro reflects water - soluble antioxidant capacity, and the oraclipo is the lipid soluble antioxidant capacity . Trolox, a water - soluble vitamin e analog, is used as the calibration standard, and the orac result is expressed as mol te / g dry weight.the ferric reducing / antioxidant power (frap) assay was performed as previously described . A validated assay for hydroxyl radical absorbance capacity (horac) was also performed according to described methods . The peroxynitrite radical averting capacity assay (norac) and superoxide radical averting capacity (sorac) assays were performed as described previously . Other antioxidant capacity assays used in the analysis of flavocoxid's antioxidant capacity include teac (trolox equivalent antioxidant capacity), a method developed by rice - evans' group and broadly applied in analyzing food samples and dpph [2,2-di(4-tert - octylphenyl)-1-picrylhydroxyl], an easy and accurate method frequently used to measure the antioxidant capacity of fruit and vegetable juices and extracts . The in vitro antioxidant activity of flavocoxid was evaluated using oxygen radical absorbance capacity (orac) procedures (brunswick laboratories, norton, mass, usa). Values are expressed as mol of trolox equivalents (te) per g of dry weight and compared to known values for vitamins c and e. the orac analysis provides a measure of the scavenging capacity of antioxidants against the peroxyl radical, which is one of the most common ros found in the body . Orachydro reflects water - soluble antioxidant capacity, and the oraclipo is the lipid soluble antioxidant capacity . Trolox, a water - soluble vitamin e analog, is used as the calibration standard, and the orac result is expressed as mol te / g dry weight . The ferric reducing / antioxidant power (frap) assay a validated assay for hydroxyl radical absorbance capacity (horac) was also performed according to described methods . The peroxynitrite radical averting capacity assay (norac) and superoxide radical averting capacity (sorac) assays were performed as described previously . Other antioxidant capacity assays used in the analysis of flavocoxid's antioxidant capacity include teac (trolox equivalent antioxidant capacity), a method developed by rice - evans' group and broadly applied in analyzing food samples and dpph [2,2-di(4-tert - octylphenyl)-1-picrylhydroxyl], an easy and accurate method frequently used to measure the antioxidant capacity of fruit and vegetable juices and extracts . Human peripheral blood mononuclear cells (pbmcs) were isolated from apheresis products (cobe laboratories, inc .) Using a histopaque density gradient and cocultured at ~4.5 10 cells in 3 ml growth medium per well in 6-well plates with lps at 10 ng / ml at 37c in a humid environment with 5% co2 . Total rnas were prepared using the qiagen rneasy kit and cdnas synthesized using the abi cdna archive kit, following the protocols of the suppliers . Qpcr assays were run in duplicate in an abi 7700 sequence detector using abi taqman gene expression primer and probe sets for cox-1 and cox-2 . Flavocoxid was compared to celecoxib, ibuprofen, and acetaminophen all at 3 g / ml for their effects on cox-1 and cox-2 expression . Cyclophilin a was used as the reference transcript for the relative quantification of rna levels to normalize gene expression . The duncan multiple range test was used to compare group means . In all cases, a probability error of less than .05 was selected as the criterion for statistical significance . Pla2 inhibitory activityother well researched flavonoids, such as green tea catechins and quercitin have been shown to inhibit pla2 thus modulating the generation of aa from membrane phospholipids [23, 24]. Little is known, however, regarding the direct inhibitory effects of either baicalin or catechin on pla2 activity . Therefore, flavocoxid was tested in a macrophage cell assay for its ability to inhibit pla2 activity.flavocoxid had a minor, nonstatistical effect on macrophage cell viability at 200 and 500 g / ml (data not shown). When flavocoxid was titrated into lps - stimulated rat peritoneal macrophage cultures, it exhibited a dose response inhibition of pla2 (ic50 = 60 g / ml) at concentrations of 50, 100, 200, and 500 g / ml significantly better than lps alone (p <.05) (figure 2). This result suggests that flavocoxid has the ability to modulate the generation of aa from membrane phospholipids produced by the destruction of tissue which occurs in oa . Other well researched flavonoids, such as green tea catechins and quercitin have been shown to inhibit pla2 thus modulating the generation of aa from membrane phospholipids [23, 24]. Little is known, however, regarding the direct inhibitory effects of either baicalin or catechin on pla2 activity . Therefore, flavocoxid was tested in a macrophage cell assay for its ability to inhibit pla2 activity . Flavocoxid had a minor, nonstatistical effect on macrophage cell viability at 200 and 500 g / ml (data not shown). When flavocoxid was titrated into lps - stimulated rat peritoneal macrophage cultures, it exhibited a dose response inhibition of pla2 (ic50 = 60 g / ml) at concentrations of 50, 100, 200, and 500 g / ml significantly better than lps alone (p <.05) (figure 2). This result suggests that flavocoxid has the ability to modulate the generation of aa from membrane phospholipids produced by the destruction of tissue which occurs in oa . Cox-1 and cox-2 cyclooxygenase inhibitory activitythe cox proteins contain two different enzymatic moieties for metabolism of aa, cyclooxygenase (co) and peroxidase (po). The co activity converts aa to pgg2 and the po activity metabolizes pgg2 to pgh2 . Subsequent platelet and cellular synthases as well as isomerases then convert pgh2 to tx as well as pgs and pcs . Medications used to treat oa, such as traditional nsaids and selective cox-2 inhibitors, block the production of pgg2, but do not affect the po site . Flavocoxid was tested for its specific co and po inhibition using purified enzymes in vitro to define its specific anti - cox-1 and cox-2 effects.flavocoxid showed no detectable anti - co cox-2 activity up to 50 g / ml demonstrated by a downturn in the curve and accumulation of oxygen in the assay (figure 3(a)). Ns-398, a strong selective cox-2 inhibitor, had a co cox-2 ic50 of 0.095 g / ml . Compared with indomethacin, which gave a co cox-1 ic50 of 0.012 g / ml, flavocoxid had an ic50 of 25 g / ml . These results suggest that flavocoxid has little anti - co activity on the cox enzymes compared to well - characterized anti - inflammatory agents . The cox proteins contain two different enzymatic moieties for metabolism of aa, cyclooxygenase (co) and peroxidase (po). The co activity converts aa to pgg2 and the po activity metabolizes pgg2 to pgh2 . Subsequent platelet and cellular synthases as well as isomerases then convert pgh2 to tx as well as pgs and pcs . Medications used to treat oa, such as traditional nsaids and selective cox-2 inhibitors, block the production of pgg2, but do not affect the po site . Flavocoxid was tested for its specific co and po inhibition using purified enzymes in vitro to define its specific anti - cox-1 and cox-2 effects . Flavocoxid showed no detectable anti - co cox-2 activity up to 50 g / ml demonstrated by a downturn in the curve and accumulation of oxygen in the assay (figure 3(a)). Ns-398, a strong selective cox-2 inhibitor, had a co cox-2 ic50 of 0.095 g / ml . Compared with indomethacin, which gave a co cox-1 ic50 of 0.012 g / ml, flavocoxid had an ic50 of 25 g / ml . These results suggest that flavocoxid has little anti - co activity on the cox enzymes compared to well - characterized anti - inflammatory agents . Cox-1 and cox-2 peroxidase inhibitory activityit has been shown that imbalances in cox-2 versus cox-1 inhibition by selective cox-2 inhibitors in the generation of various aa metabolites can contribute to edema, hypertension, and myocardial infarctions . Thus, the specially formulated, proprietary mixture of baicalin and catechin has been designed in an attempt to balance cox-1 and cox-2 metabolism of aa focusing on inhibition of the po activity of these enzymes . A cleavable, peroxide chromophore tmpd was used to assess the anti - po activity of flavocoxid on purified enzyme systems using cox-1 and cox-2.analysis of 90% pure baicalin used to formulate flavocoxid was slightly more selective against the cox-2 (ic50 = 10 g / ml) compared to cox-1 po activity (ic50 = 13 g / ml) activity (data not shown). The 90% pure catechin used in the formulation, however, showed preferential inhibitory activity towards the po of cox-1 (ic50 = 2.5 g / ml) versus cox-2 (ic50 = 15 g / ml) (data not shown). In an attempt to balance the po cox-1 with that of po cox-2 inhibition activity, baicalin and catechin flavocoxid showed relatively balanced inhibition of both cox-1 and cox-2 po activities with ic50s of 12.3 and 11.3 g / ml, respectively (figure 3(b)). These results as well as the relative lack of co activity on cox-1 and cox-2 suggest that flavocoxid exerts its effects via modulation of the po activity of these enzymes . It has been shown that imbalances in cox-2 versus cox-1 inhibition by selective cox-2 inhibitors in the generation of various aa metabolites can contribute to edema, hypertension, and myocardial infarctions . Thus, the specially formulated, proprietary mixture of baicalin and catechin has been designed in an attempt to balance cox-1 and cox-2 metabolism of aa focusing on inhibition of the po activity of these enzymes . A cleavable, peroxide chromophore tmpd was used to assess the anti - po activity of flavocoxid on purified enzyme systems using cox-1 and cox-2 . Analysis of 90% pure baicalin used to formulate flavocoxid was slightly more selective against the cox-2 (ic50 = 10 g / ml) compared to cox-1 po activity (ic50 = 13 g / ml) activity (data not shown). The 90% pure catechin used in the formulation, however, showed preferential inhibitory activity towards the po of cox-1 (ic50 = 2.5 g / ml) versus cox-2 (ic50 = 15 g / ml) (data not shown). In an attempt to balance the po cox-1 with that of po cox-2 inhibition activity, baicalin and catechin flavocoxid showed relatively balanced inhibition of both cox-1 and cox-2 po activities with ic50s of 12.3 and 11.3 g / ml, respectively (figure 3(b)). These results as well as the relative lack of co activity on cox-1 and cox-2 suggest that flavocoxid exerts its effects via modulation of the po activity of these enzymes . 5-lox inhibitory activitynsaids and cox-2 inhibitors do not inhibit the 5-lox pathway to prevent the accumulation of leukoattractive and vasoconstrictive lts . Inhibition of cox-1 and cox-2 by nsaids or selective cox-2 inhibitors has also been shown to shunt aa metabolism down the 5-lox pathway, resulting in an overabundance of these fatty acid metabolites which can adversely affect multiple organs . Increased levels of lts are associated with a variety of pathological conditions including asthma, gastric ulcerations, renal insufficiency, and cardiovascular complications [29, 30]. Cyclooxygenase enzyme inhibition has also been shown to increase the level of ltb4 in synovial fluid, perhaps inducing further damage to cartilage . Flavocoxid was titrated along with purified 5-lox enzyme in the presence of an oxygen sensing chromogen in vitro to detect the formation of unstable hpetes, an intermediate in the formation of lts.individual tests of baicalin and catechin revealed differences in 5-lox inhibitory capacity . Baicalin exhibited a relatively strong inhibition of the 5-lox enzyme with an ic50 of 65 g / ml in comparison to catechin (ic50 = 300 g / ml) (figure 3(c)). Flavocoxid, presumably reflecting the ratio of these two molecules in the formulation, inhibited the 5-lox enzyme with an ic50 of 110 g / ml . With the exception of the known 5-lox inhibitor, phenidone (ic50 = 1.3 g / ml), used as a positive control in these assays, no other nsaid or selective cox-2 inhibitors, including rofecoxib, valdecoxib, diclofenac, meloxicam, and aspirin (data not shown), showed any anti-5-lox activity . Ibuprofen with a small amount of 5-lox inhibition at the highest concentration tested (no ic50 could be determined) is also shown in comparison to celecoxib (figure 3(c)). These results suggest that flavocoxid also modulates the formation of lts from 5-lox and may prevent an accumulation of these key inflammatory factors which contribute to tissue damage through a putative 5-lox shunt seen with nsaids . Nsaids and cox-2 inhibitors do not inhibit the 5-lox pathway to prevent the accumulation of leukoattractive and vasoconstrictive lts . Inhibition of cox-1 and cox-2 by nsaids or selective cox-2 inhibitors has also been shown to shunt aa metabolism down the 5-lox pathway, resulting in an overabundance of these fatty acid metabolites which can adversely affect multiple organs . Increased levels of lts are associated with a variety of pathological conditions including asthma, gastric ulcerations, renal insufficiency, and cardiovascular complications [29, 30]. Cyclooxygenase enzyme inhibition has also been shown to increase the level of ltb4 in synovial fluid, perhaps inducing further damage to cartilage . Flavocoxid was titrated along with purified 5-lox enzyme in the presence of an oxygen sensing chromogen in vitro to detect the formation of unstable hpetes, an intermediate in the formation of lts . Baicalin exhibited a relatively strong inhibition of the 5-lox enzyme with an ic50 of 65 g / ml in comparison to catechin (ic50 = 300 g / ml) (figure 3(c)). Flavocoxid, presumably reflecting the ratio of these two molecules in the formulation, inhibited the 5-lox enzyme with an ic50 of 110 g / ml . With the exception of the known 5-lox inhibitor, phenidone (ic50 = 1.3 g / ml), used as a positive control in these assays, no other nsaid or selective cox-2 inhibitors, including rofecoxib, valdecoxib, diclofenac, meloxicam, and aspirin (data not shown), showed any anti-5-lox activity . Ibuprofen with a small amount of 5-lox inhibition at the highest concentration tested (no ic50 could be determined) is also shown in comparison to celecoxib (figure 3(c)). These results suggest that flavocoxid also modulates the formation of lts from 5-lox and may prevent an accumulation of these key inflammatory factors which contribute to tissue damage through a putative 5-lox shunt seen with nsaids . When elevated aa is present in organisms and it is exposed to ros, f2-isoprostanes and 4-hydroxynonenal (hne) are produced along with elevated malondialdehyde (mda) in the presence of epinephrine and collagen . These oxidative conversion products are elevated in the synovial fluid, synoviocytes, and serum of oa patients when compared to healthy control subjects and have been shown to stimulate the production of cartilage - degrading matrix metalloproteinases . No nsaids used in the treatment of oa possess potent antioxidant properties which could potentially damp inducible inflammation . Flavocoxid modulates the production of inflammatory cytokines and inducible nitrous oxide synthase (inos) in cell assays presumably through an antioxidant mechanism of action [5, 34], but the extent of its antioxidant capacity has never been investigated . To evaluate the antioxidant capacity of flavocoxid the oractotal for flavocoxid was found to be higher than the control antioxidants vitamin c (oractotal = 2000 molte / g) and vitamin e (oractotal = 1100 molte / g) at 3719 molte / g with the oraclipo (19 molte / g) contributing little to the overall orac score (table 1). Flavocoxid had a horac value of 1326 mol cae / g for hydroxyl radicals, which have also been implicated in joint damage . Flavocoxid, with a norac value of 1936 molte / g, has a high capacity to neutralize peroxynitrite radicals . On the contrary, superoxide radical scavenging capacity (sorac) is relatively low for flavocoxid (table 1). Shieh et al . Found that baicalein had a much higher superoxide anion capacity compared to baicalin . This may explain the relatively low sorac value of flavocoxid, as baicalin, rather than baicalein, the gut bacterial breakdown product absorbed systemically from flavocoxid, was tested in this assay . With respect to dpph, our results are consistent with others that show baicalin and epicatechin, the stereoisomer of catechin, to be strong dpph scavengers . Our results also show that flavocoxid has a high teac of 2456 molte / g (table 1). When elevated aa is present in organisms and it is exposed to ros, f2-isoprostanes and 4-hydroxynonenal (hne) are produced along with elevated malondialdehyde (mda) in the presence of epinephrine and collagen . These oxidative conversion products are elevated in the synovial fluid, synoviocytes, and serum of oa patients when compared to healthy control subjects and have been shown to stimulate the production of cartilage - degrading matrix metalloproteinases . No nsaids used in the treatment of oa possess potent antioxidant properties which could potentially damp inducible inflammation . Flavocoxid modulates the production of inflammatory cytokines and inducible nitrous oxide synthase (inos) in cell assays presumably through an antioxidant mechanism of action [5, 34], but the extent of its antioxidant capacity has never been investigated . To evaluate the antioxidant capacity of flavocoxid the oractotal for flavocoxid was found to be higher than the control antioxidants vitamin c (oractotal = 2000 molte / g) and vitamin e (oractotal = 1100 molte / g) at 3719 molte / g with the oraclipo (19 molte / g) contributing little to the overall orac score (table 1). Flavocoxid had a horac value of 1326 mol cae / g for hydroxyl radicals, which have also been implicated in joint damage . Flavocoxid, with a norac value of 1936 molte / g, has a high capacity to neutralize peroxynitrite radicals . On the contrary, superoxide radical scavenging capacity (sorac) is relatively low for flavocoxid (table 1). Shieh et al . Found that baicalein had a much higher superoxide anion capacity compared to baicalin . This may explain the relatively low sorac value of flavocoxid, as baicalin, rather than baicalein, the gut bacterial breakdown product absorbed systemically from flavocoxid, was tested in this assay . With respect to dpph, our results are consistent with others that show baicalin and epicatechin, the stereoisomer of catechin, to be strong dpph scavengers . Our results also show that flavocoxid has a high teac of 2456 molte / g (table 1). Nitrite suppression by flavocoxidstable nitrite is used as a measure of no produced from inos from synovial macrophages in oa . Nitric oxide may be involved in the destruction of proteoglycan in cartilage by inducing the production of matrix metalloproteinase . To assess flavocoxid's effect on the production of this highly reactive oxidative molecule, lps - stimulated rat peritoneal macrophages expressing higher levels of inos were used in the presence of increasing levels of the flavonoid preparation . Nitrite measured by griess reaction and subsequent spectrophotometric analysis measured flavocoxid's no damping capacity.when flavocoxid was titrated into cultures, it suppressed nitrite production in a dose - dependent manner with an ic50 of 38 g / ml compared to cultures with lps alone (p <.05) (figure 4), suggesting that flavocoxid downregulates the production of or neutralizes no directly . Flavocoxid's activity in reducing no production through damping of inos or through inactivation of no by a direct antioxidant effect could help prevent to breakdown of the proteoglycan in cartilage . Stable nitrite is used as a measure of no produced from inos from synovial macrophages in oa . Nitric oxide may be involved in the destruction of proteoglycan in cartilage by inducing the production of matrix metalloproteinase . To assess flavocoxid's effect on the production of this highly reactive oxidative molecule, lps - stimulated rat peritoneal macrophages expressing higher levels of inos were used in the presence of increasing levels of the flavonoid preparation . Nitrite measured by griess reaction and subsequent spectrophotometric analysis measured flavocoxid's no damping capacity . When flavocoxid was titrated into cultures, it suppressed nitrite production in a dose - dependent manner with an ic50 of 38 g / ml compared to cultures with lps alone (p <.05) (figure 4), suggesting that flavocoxid downregulates the production of or neutralizes no directly . Flavocoxid's activity in reducing no production through damping of inos or through inactivation of no by a direct antioxidant effect could help prevent to breakdown of the proteoglycan in cartilage . Flavocoxid effects on cox-1 and cox-2 gene expressioninducible inflammatory genes are generally up - regulated by the generation of ros which induce nfb expression and activation by release of cytosolic ib-bound nfb from the cytoplasm and subsequent translocation to the nucleus . Cytokine (il-1, il-6 and tnf) as well as cox-2, 5-lox and inos genes have an nfb binding site in their promoter regions . Cyclooxygenase-1 gene expression is not inducible, but is regulated through intron elements to yield constitutively produced protein in many cell types . Flavocoxid is known to damp inducible inflammatory gene and protein production, but has not been compared directly to other nsaids or analgesics for this activity . Therefore, flavocoxid was added to pbmcs at a fixed concentration and compared directly to celecoxib, ibuprofen, and acetaminophen for its effects on cox-1 and -2 expression.when flavocoxid was added at 3 g / ml into cultures of pbmcs stimulated with lps, there was a 20-fold down - regulation of cox-2 (figure 5), but only a 3-fold reduction in cox-1 expression . At the same concentration, celecoxib, ibuprofen, and acetominphen in fact, celecoxib and ibuprofen increased cox-2 expression 2 to 3 fold with acetaminophen exhibiting less than a doubling of gene expression . Celecoxib, ibuprofen, and acetaminophen also increased cox-1 expression 1.2-, 5-, and 8-fold, respectively (figure 5). These results suggest that flavocoxid may modulate inflammatory metabolites, such as pg, by reducing cox gene expression compared to celecoxib, ibuprofen and acetaminophen which cause increased levels of cox-1 and cox-2 expression . Inducible inflammatory genes are generally up - regulated by the generation of ros which induce nfb expression and activation by release of cytosolic ib-bound nfb from the cytoplasm and subsequent translocation to the nucleus . Cytokine (il-1, il-6 and tnf) as well as cox-2, 5-lox and inos genes have an nfb binding site in their promoter regions . Cyclooxygenase-1 gene expression is not inducible, but is regulated through intron elements to yield constitutively produced protein in many cell types . Flavocoxid is known to damp inducible inflammatory gene and protein production, but has not been compared directly to other nsaids or analgesics for this activity . Therefore, flavocoxid was added to pbmcs at a fixed concentration and compared directly to celecoxib, ibuprofen, and acetaminophen for its effects on cox-1 and -2 expression . When flavocoxid was added at 3 g / ml into cultures of pbmcs stimulated with lps, there was a 20-fold down - regulation of cox-2 (figure 5), but only a 3-fold reduction in cox-1 expression . At the same concentration, celecoxib, ibuprofen, and acetominphen did not reduce cox expression . In fact, celecoxib and ibuprofen increased cox-2 expression 2 to 3 fold with acetaminophen exhibiting less than a doubling of gene expression . Celecoxib, ibuprofen, and acetaminophen also increased cox-1 expression 1.2-, 5-, and 8-fold, respectively (figure 5). These results suggest that flavocoxid may modulate inflammatory metabolites, such as pg, by reducing cox gene expression compared to celecoxib, ibuprofen and acetaminophen which cause increased levels of cox-1 and cox-2 expression . An overabundance of aa in the general population's diet of western countries has shifted the balance of fatty acid metabolism toward an increase in proinflammatory metabolite generation via the cox and 5-lox enzymatic pathways [39, 40]. This has been accompanied by an increased generation of lipid peroxidation products by aa - oxidation as a result of poor antioxidant status . There is a strong link between metabolic defects in fatty acid metabolism or an overabundance of dietary fatty acids and the incidence of oa . For example, fatty acid levels in bone have been shown to be 5090% higher in oa patients compared to controls . Poor oxidative status, as well as the production of oxidized lipids from aa, has also been linked to chondrocyte apoptosis, activation of latent matrix metalloproteinases, and cartilage matrix degradation due to upregulation of inflammatory gene expression [38, 42]. Therefore, an anti - inflammatory agent with pleiotropic activities could help modulate these predisposing dietary factors . Kim et al . Reviewed this subject and found that a wide variety of flavonoids modulate the activities of aa metabolizing enzymes, such as pla2, cox, and 5-lox and the no producing enzyme, inos . We sought to test flavocoxid, a prescription product, to determine its impact on these pathways which contribute to oa . Lipid peroxidation, as a result of poor oxidative status, destabilizes cell membranes leading to induction of calcium - dependent pla2 which hydrolytically attacks phospholipids leading to the generation of aa . In joints, breakdown of chondrocyte cell membranes provides the substrate for pla2 conversion to aa . With their potent antioxidant capacity, flavonoids can interrupt the oxidative generation of aa from phospholipids and reduce the downstream production of inflammatory metabolites from aa metabolism, oxidative damage, and induction of inducible inflammatory pathways [34, 4649]. In our study this suggests that flavocoxid may act to limit the conversion of phospholipids from damaged cell membranes to aa upstream of the cox and 5-lox metabolic pathways . In addition, the reduction in nitrite, the stable breakdown product of no, may prevent the production of matrix metalloproteinase from macrophages present in the synovium . In support of these findings since it is also known that no levels are positively affected by increases in cytokine production, flavocoxid's cytokine - reducing effect [5, 48] may inhibit the increase in inos expression and no levels . The current experiments demonstrate that, unlike nsaids, flavocoxid does not appreciably inhibit co metabolism of aa to pgg2 . There are conflicting reports that the po and co activities are separable in the cox enzymes [51, 52]. When the po site was mutated, there were delays in the metabolite generation implying that some other cellular peroxidase may compensate for the mutated activity . Therefore, co and po activities are coupled in the cox-1 and cox-2 enzymes to efficiently produce end products . Flavocoxid was shown to downregulate both pge2 and ltb4 production in cell and animals models of inflammation [5, 34, 48, 56]. Flavocoxid did not, however, block the production of txa2 in a human platelet function study, suggesting the presence of another compensatory mechanism that bypasses its po inhibitory activity on the cox enzymes to allow for generation of the pgh2 intermediate which is then converted to txa2 . This differential mechanism of po versus co inhibition also explains flavocoxid's lack of interaction with aspirin since aspirin specifically modifies the cox-1 binding site . It has been demonstrated that patients with oa of the knee have higher than normal levels of oxidative species and antioxidant enzymes in their synovial fluid . Pure tea catechins and (+) -catechin, found in flavocoxid, are known to be strong antioxidants with high orac scores between 13,000 and 20,000 mol te / g, respectively [59, 60]. Baicalin was shown, however, to have a much lower orac score of ~365 molte / g . Although flavocoxid has a lower antioxidant capacity compared to catechin itself, the oractotal score is still approximately 3 times that of mixed tocopherols and almost twice that of vitamin c. altavilla et al . Showed that flavocoxid's antioxidant capacity reduced cellular conversion of aa to mda in the presence of epinephrine and collagen normally elevated in oa patients . Nuclear factor-b binding to a model substrate and ib protein expression was also elevated in the same experiments suggesting that flavocoxid restored the cytoplasmic inhibitory control of this ros - inducible transcription factor . An impure mixture of baicalin and catechin has also been shown to decrease oxidative species in synovial fluid in humans with oa suggesting a direct antioxidant effect on the level of ros species in the joint . Previous experiments have demonstrated that flavocoxid downregulates cox-2 and 5-lox protein expression, but not cox-1 . . Recently showed that a combination of baicalin and catechin dramatically decreases cox-2 and cytokine expression, but has only a marginal effect on cox-1 in a human cell model . We found that flavocoxid also decreases cox-2 expression by ~20-fold in human pbmcs, while having a much smaller effect on cox-1 (~3-fold). This work showed, however, that celecoxib and ibuprofen promoted noticeable increases in cox-1 and cox-2, suggesting both therapies could potentially contribute to inducible pathways of inflammation . When combined with a strong antioxidant capacity and previous findings on damping inflammatory gene expression [5, 34, 48, 63], the results presented here suggest that flavocoxid acts through an antioxidant mechanism to control inducible inflammatory gene expression in addition to modulating fatty acid processing enzymes including 5-lox . Wallace and ma suggested that metabolic shunting toward the 5-lox pathway was a major contributor to gastric injury in humans . When gastric mucosa was stimulated with calcium ionophore or arthritis patients were exposed to nsaids, there was a reduction in the release of pge2 and an increase in leukoattractive ltb4 and vasoconstrictive ltc4 and ltd4 [65, 66]. Compatible with these findings, a long - term clinical trial has shown that flavocoxid has significantly fewer upper gastrointestinal adverse events compared to naproxen [12, 67]. Similarly, a phase - iv - like postmarketing study showed that flavocoxid was well - tolerated in previously nsaid (including celecoxib) intolerant patients who ceased or decreased gastroprotective use by over 30% . Performed a cost analysis which suggests that this safety advantage would make flavocoxid less expensive compared to generic naproxen in a medicare population over a one - year time frame . A well - controlled endoscopy study is needed to definitively judge flavocoxid's gastrointestinal safety . Gambaro has suggested that dual inhibition of cox and 5-lox enzymes may also offer advantages in terms of renal toxicity, which may also affect cv complications . [12, 67] found that there were statistically fewer incidences of edema for flavocoxid compared to naproxen . Though not reaching statistical significance, creatinine levels trended lower in the flavocoxid group and higher in the naproxen group . Specific well - controlled studies in renal compromised subjects are needed to truly assess flavocoxid's safety in these patients . Flavocoxid is the only currently marketed prescription anti - inflammatory agent that modulates cox enzymes via an anti - po activity, inhibits 5-lox - mediated lt production, and has a broad, strong antioxidant activity which downregulates inducible inflammatory gene expression as well as neutralizes ros thereby preventing the conversion of aa to oxidized lipids . Flavocoxid's putative mechanism of action is shown in figure 6 and is presumed to be the reason for the favorable safety profile seen in clinical trials and postmarketing surveillance . Work is currently underway to determine if this broad - based mechanism of action can preserve cartilage structure.
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Neovascular age - related macular degeneration (namd) is the leading cause of visual loss among the elderly population in developed countries [1, 2]. Before the introduction of intravitreal antivascular endothelial growth factor therapy for namd, only prevention for visual loss might have been achieved in a limited number of patients with different treatment options like laser photocoagulation and photodynamic therapy [37]. The introduction of bevacizumab (full length antibody against vegf - a) and ranibizumab (fab part of antibody against vegf - a) has led the vast majority of the patients to prevent the baseline visual acuity [816]. The pivotal multicenter studies with ranibizumab, like marina, anchor, pronto, excite, and catt, showed that ranibizumab is effective to prevent baseline visual acuity in up to 95% of the patients and is effective to make an improvement in visual acuity in up to 40% of the patients [913]. Monthly ranibizumab treatment for namd was found to be effective in marina and anchor studies; however, an attempt then aroused to decrease the injection numbers . Therefore, studies like pronto and fusion were designed to evaluate the effectiveness of as - needed treatment regimens, and ranibizumab was found to be effective in the treatment of namd on as - needed treatment regimens [11, 14]. Catt study was designed to compare the outcomes of monthly and as - needed treatment regimens for both bevacizumab and ranibizumab . This study revealed that the visual acuity outcomes were similar for monthly bevacizumab versus monthly ranibizumab, monthly bevacizumab versus as - needed bevacizumab, and monthly ranibizumab versus as - needed ranibizumab . Only monthly ranibizumab was found to be more effective than as - needed bevacizumab . Optical coherence tomography (oct) is a noninvasive diagnostic device that provides cross - sectional images of the retina like ultrasound [17, 18]. Time - domain oct systems have a scan rate of 400 a - scans per second which allow an axial resolution of 810 microns, and spectral - domain oct systems have a scan rate of 20.00065.000 a - scans per second which allow an axial resolution of 37 microns and provide a three - dimensional view of the retina . Although fluorescence angiography is the gold standard for the diagnosis and followup of retinal vascular diseases and namd for several decades, it was largely replaced by oct after the introduction of oct - guided, flexible treatment regimens for namd with anti - vegf agents [17, 18]. In this study, we aimed to evaluate the activity of choroidal neovascularization with sd - oct in the namd patients who were on an as - needed treatment regimen with ranibizumab and were diagnosed to have an inactive choroidal neovascularization with td - oct and clinical examination one month after intravitreal ranibizumab injection . This prospective, comparative, cross - sectional study included 49 eyes of 49 consecutive namd patients who were on an as - needed treatment regimen with intravitreal ranibizumab (ivr). The patients underwent ivr injections at beyoglu eye training and research hospital between march 2012 and april 2012 . Approval for data collection and analysis was obtained from the ethics committee of the hospital, and written informed consent was provided from all patients . The methodology of the study was designed in accordance with the tenets of the helsinki declaration . The treatment regimen was as follows: for the first three months of treatment, all patients received monthly doses of ranibizumab 0.5 mg/0.05 ml . The patients were then examined monthly and were retreated if they met any of the following criteria: visual loss of 1 or more lines, new perimacular hemorrhage, evidence of cnv enlargement on examination or fluorescein angiography, any amount of persistent subretinal, intraretinal, or subretinal pigment epithelial (subrpe) fluid one month after an injection.the patients who had a diagnosis of namd, were currently on an as - needed treatment regimen with ivr, underwent an ivr injection a month ago, did not experience visual loss since the last visit, did not show any activity of cnv on fundus examination, and did not show any amount of subretinal, intraretinal, or subretinal pigment epithelium (subrpe) fluid on td - oct were included in the study . The patients were not included in the study if the quality of the oct scans were not sufficient with any of the td - oct or sd - oct devices . Visual loss of 1 or more lines, new perimacular hemorrhage, evidence of cnv enlargement on examination or fluorescein angiography, any amount of persistent subretinal, intraretinal, or subretinal pigment epithelial (subrpe) fluid one month after an injection . Data collected from the patients' records included age, gender, central retinal thickness defined by td - oct and sd - oct, the fluid type revealed by sd - oct, and the vitreoretinal surface abnormalities revealed by td - oct and sd - oct . All patients underwent a standardized examination including measurement of bcva via the early treatment diabetic retinopathy study (etdrs) chart at 4 meters, slit - lamp biomicroscopy and fundus examination, and measurement of intraocular pressure (iop) via applanation tonometry . Fundus photography, and optical coherence tomography (oct) imaging with td - oct (stratus oct tm; carl zeiss meditec inc ., dublin, ca, usa) and sd - oct (spectralis; heidelberg engineering, heidelberg, germany) were performed . Time - domain oct imaging was performed via stratus oct 3000 (carl zeiss, meditec inc ., dublin, ca, usa). Fast macular thickness map (fastmac) protocol was used to obtain the retinal scans, within a scan time of 1.9 seconds, which acquires six evenly spaced 6-millimeter (mm) radial lines, each consisting of 128 a scans per line, intersecting at the fovea (total of 768 sampled points). The crt was defined as the distance between the internal limiting membrane (ilm) and the photoreceptor junction of the inner and outer segments via the automatic analysis package of stratus oct . Spectral - domain oct imaging was performed via spectralis oct (heidelberg engineering, heidelberg, germany). Fast macula protocol was used to obtain the retinal scan, with a automatic real time (art) mean value of 9, which acquires 25 horizontal lines (6 6 mm area), each consisting of 1024 a scans per line . The crt was defined as the distance between the ilm to the outer border of the retinal pigment epithelium via the automatic segmentation algorithms of the spectralis software . The main outcome measure of the study was the rate of the patients who had any amount of subretinal, intraretinal, or subrpe fluid on sd - oct and who were diagnosed as inactive after clinical examination and td - oct imaging . The secondary outcome measures were the crt values defined via the two devices and the ability of detecting vitreoretinal surface disorders via the two devices . For the statistical analyses, the mean (sd) of the differences was calculated . Independent sample t - test was used to compare continuous parameters between the two devices, and chi - square test was used for nominal parameters . Statistical analyses were made using commercially available software spss version 16.0 (spss inc ., chicago, il). 29 patients (59.2%) were female and 20 patients (40.8%) were male . Mean crt of the patients via td - oct was 218.1 51.3 microns (range 139418 microns), and the mean crt via sd - oct was 325.7 78.8 (range 222508 microns). The mean ratio between the mean crt measured with sd - oct and crt measured with td - oct was 1.51 0.09 . As the patients who did not show any amount of subretinal, intraretinal, or subrpe fluid in td - oct were the subjects of this study, any kind of retinal fluid was not detected in td - oct . However, 16 of the 49 patients (32.6%) showed subretinal, and/or intraretinal, and/or subrpe fluid on sd - oct . In detail, 8 patients showed (16.3%) subretinal fluid, 10 patients (20.4%) showed intraretinal fluid, and 3 patients (6.1%) showed subrpe fluid (table 1) (figure 1). The ability of the two devices in detecting vitreoretinal surface abnormalities was summarized in table 2 . Since the introduction of the sd - oct, the resolution of retinal scans and parallelly our diagnostic ability increased . Many studies compared the td - oct and sd - oct systems in regard to the fluid detection, and retinal thickness measurements [1723]. In previous studies the, oct devices were usually compared in patients having had namd, diabetic macular edema, and retinal vein occlusion [1723]. However, these studies mostly evaluated the ability of these devices in detecting subretinal, intraretinal, or subrpe fluid and compared the crt measurements between them [1721]. In this study we delineated the namd patients who were diagnosed to have an inactive cnv via the td - oct and clinical examination, and then we evaluated them with the sd - oct in order to detect the additional sensitivity provided via the latter device . The additional sensitivity of the sd - oct was 32.6% in regard to cnv activity . In a study by kakinoki et al ., the macular thickness in diabetic macular edema measured with td - oct and sd - oct were compared . The macular thickness values were well correlated; however, the mean macular thickness detected with the sd - oct was reported to be 45 microns thicker than the td - oct . Compared two different sd - oct devices with td - oct in patients with macular edema secondary to diabetic retinopathy and retinal vein occlusion . The macular thickness values detected with both sd - oct devices were also found to be higher than the values detected with td - oct . Eriksson et al . Showed that sd - oct increased the repeatability and decreased variability in namd patients, and demonstrated a significant advantage of sd - oct over td - oct . Krebs et al . Mentioned the difference of crt values detected with the td - oct and the sd - oct devices and attributed this variability to the different posterior reference lines of the devices . They advised avoiding using different devices during an ongoing study and suggested a formula for comparing the values defined by the two devices . In a case, sd - oct was found to be superior to td - oct in regard to fluid and cyst detection . Querques et al . Compared the ability of the 2 sd - oct and td - oct devices in detecting the disease activity in the namd patients . Cirrus sd - oct (cirrus hd - oct, carl zeiss - meditec, inc ., dublin, ca) and spectralis sd - oct were found to be superior to td - oct especially in detecting srf and ped . They also reported that the correcting factor should be 1.48 to extrapolate the crt measurements from stratus td - oct to spectralis sd - oct, which was comparable with our value of 1.51 . Cukras et al . Reported that cirrus sd - oct was able to detect the presence of the exudative activity (subretinal or intraretinal fluid) in 48% of the namd patients who were defined as inactive with stratus td - oct . Compared 4 different sd - oct devices with stratus td - oct in regard to disease activity in the namd patients after intravitreal ranibizumab treatment . They reported that all 4 sd - oct devices were superior to td - oct in detecting subretinal fluid in three - dimensional cube mode . However, in linear b - scan mode, only the spectralis sd - oct was reported to be superior to the stratus td - oct in detecting subretinal fluid, and the oct-1000 was reported to be superior in detecting sub - rpe fluid . In addition, the presence of subretinal fluid, intraretinal cysts, and subrpe fluid on the spectralis sd - oct was reported to be 16%, 11%, and 13%, respectively, when td - oct did not show any sign of disease activity . Only the rate of the sub - rpe fluid detection of our study was lower than that of sayanagi et al . The va loss during the as - needed treatment regimens for the namd which is usually attributed to the treatment delays, the low injection numbers, the presence of ped at the baseline, and the crt fluctuations during the treatment is usually irreversible [2529]. Except one, three of these factors, treatment delays, crt fluctuations, and low injection numbers are closely related with our ability to detect disease activity . Therefore, in the era of the sd - oct devices, the td - oct seems inaccurate and insufficient in deciding whether or not retreatment is necessary at the monthly visits of the namd patients . On the other hand, while the crt is an important prognostic factor for the diabetic macular edema and retinal vein occlusion patients, and td - oct is comparable with the sd - oct devices in this group of patients, td - oct may still have a role in our retreatment decisions . Limitations of this study include the relatively small number of patients and the one - sided assessment between the two devices (we did not evaluate the nonactive patients via the sd - oct with td - oct). Strengths of this study were the prospective design and the homogeneity of the patients . In conclusion, the previous studies and the present study suggest that sd - oct is essential in the followup of the namd patients who are under an as - needed treatment regimen . Using td - oct may result in worse visual outcomes due to the treatment delays and inadequate treatment.
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Kazanjian (1938) classified tmj ankylosis, according to the site involved, into true (intracapsular) and false / pseudoankylosis (extracapsular). While true ankylosis refers to fibrous or bony ankylosis that occurs between the condylar head of the mandible and the mandibular fossa of the temporal bone, false / pseudo ankylosis refers to restriction of mandibular movement that occurs as result of pathology or physical obstruction that is outside the temporomandibular (tm) joint.pseudo-ankylosis is less common than true ankylosis . Radiographic examination is a vital diagnostic tool for the diagnosis and management of tmj ankylosis . Although conventional radiographs such as orthopantomogram (opt) and tm joint tomography have been in use, imaging techniques such as computed tomographic (ct) scanning with three - dimensional reconstruction have been developed and improved . Any pathology that afflicts the tm joint and restricts the mouth opening carries a mental stigma that overweighs the physical disability posed by the problem in growing children in reviewing the types and patterns of disruption in tm joint function, we describe a distinct case of pseudo - ankylosis of a low condylar fracture, anteriorly displaced, dislocated, and fused to the zygomatic arch, in an 8-year - old child that sustained an old trauma; the probable mechanism of injury that lead to this unique presentation is also proposed . In addition, the importance of therapy and its implications in tm joint ankylosis are briefly discussed . An 8 year - old boy, of moderate build and nourishment, accompanied by his father, presented to the department of oral / maxillofacial surgery with reduced mouth opening . Further questioning revealed that two years earlier the boy allegedly met with a road traffic accident (rta) the victim fell flat on his face, chin first, after being hit on the head, while traveling home in an auto rickshaw (a public transport vehicle commonly used in india). At the time, he sustained lacerations to his forehead and chin . A little later, the boy developed mild swelling in the right preauricular region, which eventually resolved without any form of intervention . No history of loss of consciousness or bleeding from the ear or nose was reported . Primary care constituted wound debridement and closure of the forehead laceration at a local primary health centre . As time went by, the boy s father began noticing a progressive reduction in his son s ability to open his mouth (fig ., the patient appeared to have limited mouth - opening his maximal interincisal distance measured 15 mm, and the chin was mildly retruded . Conventional imaging included an orthopantomogram (opt) that revealed deformed condylar head on the right side and shortened condyle on the left side which is outside the glenoid fossa . A unique, radiopaque bone - like projection (red arrow) situated in the right sigmoid notch between the condyle and coronoid process 2). For further elucidation, and owing to considerable distortion in regional osseous anatomy, computed tomography (ct) with 3d reconstruction was advised . The 156-slice scan revealed an osseous mass fused to the sigmoid notch between the condyle and coronoid process on the right side . Bilaterally, the condyles appeared to be deformed and shortened, reminiscent of an old fracture (fig . 4) axial views survey revealed an osseous mass located just medial to the zygomatic arch (fig . There is also fractured anteriorly dislocated condylar stump into the sigmoid notch on the right side . There is also an evidence of an osseous mass on the left side suggestive of a medially displaced fractured condyle . Taken together, a final diagnosis of post - traumatic, pseudo - ankylosis of the tm joint was made, for which surgery was planned . Routine blood investigations revealed anemia hb-(9.8gm%), nevertheless, the patient was taken up for surgery . Under nasotracheal, hypotensive anesthesia, the right tm joint was exposed via a preauricular incision the joint had no structural abnormality, however, a bony projection (seen as projection on the opt & ct scan) from the sigmoid notch loosely ankylosed (fibrous) just medial to the ipsilateral zygomatic arch was seen . Release of this ankylotic mass was done using a fissure bur, freeing it of all bony and fibrous adhesions (fig ., a 32 mm mouth - opening was successfully achieved using a hister s jaw stretcher . To further optimize the intraoperative result, an ipsilateral coronoidectomy was performed so as to facilitate adequate mouth - opening . Primary closure of the incision was done, and patient reversal and recovery were uneventful . The fibrous ankylotic mass was sent for histopathological analysis which revealed proliferating connective tissue with fibroblast transition to osteoblasts and areas of cartilage, osteoid and bone . Active physiotherapy was instituted on the 2nd post - operative day, and a maximum interincisal opening of 37 mm was achieved and maintained using a therabite appliance (fig . 7). An 8 year - old boy, of moderate build and nourishment, accompanied by his father, presented to the department of oral / maxillofacial surgery with reduced mouth opening . Further questioning revealed that two years earlier the boy allegedly met with a road traffic accident (rta) the victim fell flat on his face, chin first, after being hit on the head, while traveling home in an auto rickshaw (a public transport vehicle commonly used in india). At the time, he sustained lacerations to his forehead and chin . A little later, the boy developed mild swelling in the right preauricular region, which eventually resolved without any form of intervention . No history of loss of consciousness or bleeding from the ear or nose was reported . Primary care constituted wound debridement and closure of the forehead laceration at a local primary health centre . As time went by, the boy s father began noticing a progressive reduction in his son s ability to open his mouth (fig . 1). Clinically, the patient appeared to have limited mouth - opening his maximal interincisal distance measured 15 mm, and the chin was mildly retruded . Conventional imaging included an orthopantomogram (opt) that revealed deformed condylar head on the right side and shortened condyle on the left side which is outside the glenoid fossa . A unique, radiopaque bone - like projection (red arrow) situated in the right sigmoid notch between the condyle and coronoid process 2). For further elucidation, and owing to considerable distortion in regional osseous anatomy, computed tomography (ct) with 3d reconstruction was advised . The 156-slice scan revealed an osseous mass fused to the sigmoid notch between the condyle and coronoid process on the right side . Bilaterally, the condyles appeared to be deformed and shortened, reminiscent of an old fracture (fig . 4) axial views survey revealed an osseous mass located just medial to the zygomatic arch (fig . There is also fractured anteriorly dislocated condylar stump into the sigmoid notch on the right side . There is also an evidence of an osseous mass on the left side suggestive of a medially displaced fractured condyle . Taken together, a final diagnosis of post - traumatic, pseudo - ankylosis of the tm joint was made, for which surgery was planned . Routine blood investigations revealed anemia hb-(9.8gm%), nevertheless, the patient was taken up for surgery . Under nasotracheal, hypotensive anesthesia, the right tm joint was exposed via a preauricular incision the joint had no structural abnormality, however, a bony projection (seen as projection on the opt & ct scan) from the sigmoid notch loosely ankylosed (fibrous) just medial to the ipsilateral zygomatic arch was seen . Release of this ankylotic mass was done using a fissure bur, freeing it of all bony and fibrous adhesions (fig ., a 32 mm mouth - opening was successfully achieved using a hister s jaw stretcher . To further optimize the intraoperative result, an ipsilateral coronoidectomy was performed so as to facilitate adequate mouth - opening . Primary closure of the incision was done, and patient reversal and recovery were uneventful . The fibrous ankylotic mass was sent for histopathological analysis which revealed proliferating connective tissue with fibroblast transition to osteoblasts and areas of cartilage, osteoid and bone . Active physiotherapy was instituted on the 2nd post - operative day, and a maximum interincisal opening of 37 mm was achieved and maintained using a therabite appliance (fig . 7). Several case reports,, cite falls as a cause of condylar fractures in children below the age of 6 years, with intracapsular or burst fractures being more common than extra capsular fractures . However, in the current case, the patient had sustained trauma at the age of 6 years, and both the patient s condyles had a low (extracapsular) fracture . While a vast majority of mandibular condylar fractures are displaced in an anteromedial or anterolateral direction, very few cases report of a superolateral dislocation / displacement, or traumatic impaction - dislocations into middle cranial fossa with the condyle intact . However, a case of anterior dislocation and displacement of a fractured condyle in a child below 6 years of age is rarely seen . The probable mechanism explained in the literature for the displacement of the fractured condylar segment has been attributed to the action of unopposed pull of lateral pterygoid muscle on the fractured condylar segment leading to anteromedial displacement . In the present case scenario the probable explanation that could be given for unusual anterior displacement and dislocation of the fractured condylar segment against the usual action of lateral pterygoid is as follows:1stripping of lateral pterygoid due to the impact of the injury, failing the action of lateral pterygoid on the fractured condyle.2open mouth position during the impact causing the fracture of the condyle in a position anterior to the eminence with resultant premature displacement of the residual major component of the mandible into the glenoid fossa preventing any further movement of the already anteriorly displaced fractured condylar segment towards either lateral or medial or superior directions . Stripping of lateral pterygoid due to the impact of the injury, failing the action of lateral pterygoid on the fractured condyle . Open mouth position during the impact causing the fracture of the condyle in a position anterior to the eminence with resultant premature displacement of the residual major component of the mandible into the glenoid fossa preventing any further movement of the already anteriorly displaced fractured condylar segment towards either lateral or medial or superior directions . The patient revealed that treatment had been delayed owing to poor socioeconomic status, and late reporting / referral to doctors, undesirably translating into a prolonged period of restricted mouth opening due to pain following trauma, eventually leading to psuedo - ankylosis . Miyamoto stated that restricted jaw movement is not the determinant factor, but rather the promoting agent for ankylosis . Malnourishment could be another factor influencing such an outcome, as recent evidence indicates that, in malnutrition, impaired callus formation as well as fibrous ankylosis in the tm joint result on healing of a displaced condylar process . Complete remodeling of the temporomandibular joint bilaterally, was evident on the ct scan, the findings of which are consistent with those of lindalh, who reports that 20 of 27 study patients, in the age group of 311 years, had complete return to normal skeletal relationship, a finding that was not observed in other age groups . In our case, conventional radiographs revealed altered regional (osseous) anatomy, further supplemented by a 3d ct scan to locate the condylar fragment, displaced and/or dislocated into the sigmoid notch, and study its relationship to the zygomatic arch, as the latter modality greatly increases diagnostic accuracy in tmj ankylosis imaging . Cases of condylar fractures are common in both developed and developing countries, but cases of post - traumatic tm joint ankylosis are few in developed countries compared to developing countries for various reasons as stated by dongmei he et al . & gururaj arakeri et al ., . Tm joint ankylosis may be very recent or old but of vital importance to any kind is an accurate diagnosis for successful management outcomes . It is equally important to recapitulate the types and patterns of disruption in tm joint function when setting out to open these cases as most cases usually get away without open treatment, not to mention the considerable physical and psychological insult otherwise associated with tm joint ankylosis and its management . Institution of aggressive physical therapy as early as possible is as important as the surgical intervention itself, if not more . Complications, though rare, are potentially fatal . To conclude, we herein report a unique case of anterior dislocation of fractured mandibular condyle leading to pseudo - ankylosis in a 8 yr old child . In this case report this case report also suggests that unusual dislocations of mandibular condyle do occur in children, which require careful planning and treatment . Written informed consent was obtained from the patient for publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . Dr ranjit kumar p: treated the patient, writing the paper and submission, dr naveen g: obtaining patient records, followed up the patient, dr raja satish: assisting in treating the patient, writing the paper, dr srinivas chakravarthy p: treated the patient, review the article, dr l krishna prasad: assisted in reivising the article.
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Spontaneous tumors of the rat submandibular gland are very rare, 1, 2 and most of them are diagnosed as adenomas or adenocarcinomas originating from acinar cells and ductal epithelia . 3 to our knowledge, there are few reports on detailed analyses of tumors in the submandibular glands of rats . In the present case, the results of immunohistochemical and ultrastructural observations indicated that the rat submandibular gland tumor was probably derived from basal cells located in the duct . The animal was a 10-week - old female galas rat (brlhan: wist@jcl, purchased from clea japan, inc ., tokyo, japan) that was assigned to the control group in a 4-week repeated - dose oral toxicity study and subjected to the final sacrifice according to the experimental protocol . During the treatment period, there was no abnormality in clinical signs or body weight of the animal . At necropsy, a white nodule (about 5 3 mm in size) was found in the right submandibular gland . In other organs the animal was treated in accordance with the rules for approval of animal experiments in sugi institute of biological science co., ltd . The submandibular gland was routinely fixed in 10% phosphate - buffered formalin, and embedded in paraffin and sectioned . Immunohistochemical staining was performed using the following primary antibodies: cytokeratin (ae1/ae3; 1:20, dako, denmark), p63 (1:50, dako, denmark), alpha - smooth muscle actin (sma) (1:50, dako, denmark), vimentin (1:50, dako, denmark), desmin (1:100, dako, denmark), s-100 (1:400, lvc, usa) and proliferating cell nuclear antigen (pcna; 1:100, dako, denmark). The deparaffinized sections were incubated in the antibodies at room temperature for 1 hr and then in the envison+system - hrp labeled polymer (dako, denmark) at room temperature for 30 min, and the staining was visualized with diaminobenzidine . For transmission electron microscopic examinations, the tissue kept in 10% phosphate - buffered formalin was postfixed in 1% osmium, routinely processed to epoxy resin - embedded ultrathin sections and stained with uranyl acetate and lead citrate . Histopathologically, oval to spindle - shaped and pale basophilic tumor cells proliferated closely and formed variably sized foci or grew diffusely in the submandibular gland (figs . The nodule separated by thin interstitial connective tissue and lobular structures were partially formed (fig . The border between the nodule and surrounding normal tissue was mainly clear, and the nodule partially spread into or invaded the surrounding normal tissue . The nuclei of the tumor cells showed various sizes and many mitotic figures and contained poor chromatin and one to a few nucleoli (fig . Ductal and acinar structures, cribriform patterns and keratinization were not found in the tumor . Immunohistochemically, the tumor cells showed a diffusely positive reaction for p63 in the nuclei (fig . 2) and for cytokeratin (ae1/ae3) in the cytoplasm . There was a negative reaction for sma, vimentin, desmin and s-100 . Ultrastructurally, the tumor cells contained many tonofilaments in their cytoplasm and a few desmosomes at the intercellular portion (fig . The anti - p63 protein is a selective marker for both basal and myoepithelial cells, 4, 5 and the anti-sma protein is a selective marker for myoepithelial cells . In addition, the reliable identification method of myoepithelial cells is thought to be an immunohistochemical method using anti-sma protein, together with anti - vimentin protein or anti - s-100 protein . 6, 7 in the present case, the nuclei of the tumor cells were positive for anti - p63 protein, whereas no tumor cells showed a positive reaction for anti-sma protein . Furthermore, the tumor cells were positive for cytokeratin (ae1/ae3) and negative for vimentin, s-100 and desmin . Based on the results of immunohistochemical examinations, the tumor cells were considered to be derived from basal cells . Ultrastructurally, myoepithelial cells generally possess myofilaments with dense bodies . 8 in the present case, the tumor was not considered to originate from myoepithelial cells because there were no myofilaments in the cytoplasm . Since the tumor cells had many mitotic figures, a positive reaction to anti - pcna protein, and partially necrotic foci and infiltrated into the surrounding normal tissue, the tumor was regarded as a malignant tumor . Based on these results, the tumor was diagnosed as a spontaneous basal cell carcinoma . In the normal submandibular gland, basal cells are located in the excretory duct 3; however, tonofilaments, the existence of which is one of the characteristic features of squamous cells, were observed in the cytoplasm of the tumor cells . Squamous cells are normally located in the end portion of the excretory duct in the submandibular gland . 3, 9 therefore, the tumor is considered to have originated from the basal cells in the excretory duct near the opening to the oral cavity . Adenocarcinoma because this tumor did not clearly show the ductal structure . In humans, tumors of the submandibular gland originating from basal cells basal cell adenocarcinomas. The histologic appearance of the basal cell adenoma is dominated by relatively uniform, monomorphous proliferation of basaloid cells . While each tumor generally has a uniform histomorphologic architecture, variations among tumors allow categorization into solid, trabecular, tubular and membranous types . Basal cell adenocarcinoma is very similar to basal cell adenoma cytologically and histomorphologically and manifests infiltrative growth and incidental metastasis . 9 in rats, however, there are no reports on the classification of basal cell adenomas or adenocarcinomas in the submandibular gland.
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Presenteeism is defined as the problem of workers being on the job, but, because of illness or other medical conditions, not fully functioning1 . While many employers have always been aware of costs due to absenteeism, there is now increasing evidence that the presence of ill or medically impaired employees may also result in significant costs to the organization in the form of decreased productivity while at work25 . Research on the impact of presenteeism is mainly focused on productivity loss due to chronic conditions such as allergies, arthritis, depression, and diabetes6 . Acute infectious illnesses pose an additional risk from presenteeism as employees can serve as a vector of disease transmission . To our knowledge, few studies have reported this relationship between presenteeism and infectious disease transmission in health care settings . We describe a viral gastroenteritis outbreak in a long - term care facility in which presenteeism was a key risk factor in disease transmission and extension of the outbreak . We review the risks and underlying factors that promote presenteeism, and propose novel solutions in light of its importance to public health, the safety of our health care workforce, and the health of our patients . On january 19, 2005 (day 1), three nursing home residents and one staff member at a 100-bed, two - floor urban facility developed symptoms of nausea, vomiting, and diarrhea (fig . 1). General infection control measures were reinforced, including hand hygiene education for nursing home residents and staff, contact isolation for symptomatic residents, and new surface disinfection procedures . On days 2 and 3 of the outbreak, two of these staff members reported diarrhea after arriving at work and were asked to go home after discussions with the infection control team . At this point, the public health department was notified and more restrictive measures were instituted, including closure of the dining room, suspension of group activities and outings, limitation of visitors, volunteers, and trainees, rescheduling of elective surgery and non - urgent clinic appointments, and discontinuation of new admissions . Staffing strategies were also temporarily changed so that nursing staff did not float in or out of the unit . As per policy, supervisors were instructed to refer employees with signs or symptoms of an infectious illness to employee health for diagnosis and determination of suitability to continue work . However, no daily systematic screening process took place to identify ill staff members at the start of their shift . Figure 1nursing home residents and staff with abrupt onset of nausea, vomiting, and diarrhea . Nursing home residents and staff with abrupt onset of nausea, vomiting, and diarrhea . Over the course of the next 10 days, 23 residents and 18 staff developed symptoms of nausea, vomiting, and diarrhea . Laboratory studies of affected staff and residents confirmed norovirus genotype 2 . By day 8 of the outbreak, it became increasingly clear that ill staff members continued to work despite strong recommendations to the contrary by management . Often, symptoms were not reported until employees had arrived for and sometimes completed their shifts . Infection control responded by contacting each ill staff member to verify symptoms, provide education, and ask that they remain home . Several nursing staff members who were symptomatic at work were asked to leave as soon as they reported symptoms and to not return until they received clearance from employee health . Staffing was managed through the use of registry or per diem nursing coverage when appropriate . However, on day 18, a staff member arrived at work ill with gastrointestinal symptoms . On day 21, an additional two residents developed gastroenteritis . As voluntary measures to prevent presenteeism failed, the local department of public health mandated enforcement of back to work rules . These rules required employees with gastrointestinal symptoms to obtain clearance from employee health before being allowed to return to work . This clearance was given only after 48 symptom - free hours had elapsed . The final case was identified 24 days into the outbreak, and gastroenteritis - specific infection control measures were discontinued on day 34 . The concept of presenteeism has been a topic of discussion since the 1980s in the social science and business literature worldwide1,7 . This literature focuses mainly on the economic impact based on loss of worker productivity, which has been estimated to cost more than $150 billion per year in the united states1 . Presenteeism has not yet made a significant entrance into the health care literature, unlike its counterpart absenteeism, which has received far more attention . In april 2010, a search for the term absenteeism in pubmed resulted in 5,636 english - language citations . Fifty percent of the citations were found in occupational medicine or health care management journals . The majority of these presenteeism articles focused on the impact of decreased worker productivity in workers with chronic physical or mental health conditions, or the validation of instruments designed to measure presenteeism . Only 12 of the 135 articles included workers in their study populations, and only 2 studies suggested that presenteeism might be linked to communicable disease spread8,9 . Health care personnel who return to work despite having ongoing symptoms of an infectious disease extend the risks of presenteeism far beyond reduced productivity issues into the realms of patient safety and public health . The norovirus outbreak at our long - term care facility is an example of how workers continue to work with symptoms of a contagious disease . Although we cannot directly link transmission from staff to residents due to limitations in data collection, there are many case reports that implicate ill health care workers as vectors for transmission of diseases such as influenza10, pertussis11, methicillin - resistant staphyloccous aureus12, and norovirus13,14 . Noroviruses are a leading cause of gastroenteritis outbreaks as the virus can easily spread from person to person, with as few as ten virus particles thought sufficient to cause an infection1517 . The highly contagious nature of norovirus frequently leads to outbreaks in long - term care settings as residents are often functionally dependent and require close contact with nursing staff . This dependency on staff increases the risk of staff - to - resident transmission in propagating outbreaks13,18,19 . Nursing staff may also work at more than one facility, as was the case in our reported outbreak, making presenteeism in the face of an ongoing infectious disease outbreak an even greater public health risk . The risks of an otherwise self - limited viral infection in a frail institutionalized population cannot be over - emphasized; nursing home residents in the united states are four times more likely to die from gastroenteritis than community dwelling adults20 . Health care workers as a group are very likely to continue to work when infected with diseases such as influenza and norovirus despite the serious public health risks of presenteeism21,22 . Surveyed 1,015 norwegian physicians . During 1 year, 80% of the physicians went to work during an illness for which they would have " sick listed " a patient . Two thirds of these episodes involved a possible contagious disease such as respiratory or gastrointestinal infections23 . In another survey, 1,339 uk physicians were asked if they would take the day off if they had symptoms of a severe cold . Eighty - seven percent of general practitioners and 57.8% of hospital consultants responded definitely not versus 3l.9% of the comparison group, salaried office workers24 . Ohrt and mckinney reported in a study on influenza vaccination that 70% of medical residents and students admitted that they had worked while experiencing influenza - like symptoms26 . Similarly, long - term care employees have high rates of presenteeism . In a large swedish population study27, nursing home aides were among those workers at the highest risk for working while ill . Another study done during an investigation of an acute gastroenteritis outbreak in a nursing home revealed that 94% of surveyed ill employees went to work while ill and 8% vomited while at work28 . The answer to this question is complex as it may depend upon the discipline of the employee, their social status within an organization, their level of job and financial security, and the care demands at work . Social status within an organization seems to play a large role independent of clinical discipline . For instance, a survey of physicians in various levels of training showed that medical students often cited factors that were dependent on the opinions and impressions of others as a reason to come to work with a respiratory tract infection25 . This was opposed to staff physicians who were more concerned about the delivery of patient care if they were to call in sick . A similar finding is seen within nursing . In a qualitative study of australian nurses, a sense of tension arose for nurses when determining whether to come to work while ill29 . This tension was created by previous interactions with supervisors who questioned the legitimacy of prior illnesses and focused on the need to report illnesses in a time frame that would ensure the adequacy of replacement staffing . There also may be significant financial incentives for going to work while ill for those lower in an organization s hierarchy due to a lack of paid sick leave or high levels of job insecurity30,31 . Lastly, rates of presenteeism can be influenced by care demands at work . In nordic elder care staff, higher levels of work - related demands due to understaffing and increased time pressures were associated with relatively large increases in presenteeism8 . Any recommendation to limit presenteeism's role in disease transmission even though there are no clinical trials evaluating whether time off from work while ill limits infectious disease transmission, there are some case reports that support that it may32,33 . In one retrospective study of several norovirus outbreaks in berlin, the duration of outbreaks was found to be inversely correlated with the length of time staff members took off from work33 . We were unable to identify precise reasons why employees decided not to take time off while ill in our reported outbreak . However, applying concepts from existing literature to our experience with this outbreak, we can make three health care policy recommendations with regards to presenteeism, which may improve patient safety . The first recommendation is to ensure the availability of unrestricted paid sick leave for all employees working in a health care setting in order to decrease the financial pressure to return to work when ill . A case - cohort study of new york state nursing homes indicated that homes with paid employee sick leave were less likely to have communicable disease outbreaks34 . Unfortunately, one third of american workers do not have benefits that include sick leave . Data from the national compensation survey (ncs) of the us bureau of labor statistics reveal that 12% of hospital workers and 22% of registered nurses do not have paid sick leave35 . This lack of paid sick leave can lead to a fear of losing income or employment if an employee follows public health guidelines, as seen in a related study looking at compliance with pandemic flu mitigation recommendations31 . Restrictions on paid sick leave, such as requiring a medical certificate to validate an employee's illness, can limit its effectiveness in preventing presenteeism25 . Relaxing these restrictions, as was done in the case institution by giving employees 3 days of paid sick leave without a medical certificate, can lead to employees working less often while sick without a subsequent increase in sickness absence36 . The availability of unrestricted paid sick leave is only the first step in limiting the impact of presenteeism . The norovirus outbreak at our long - term care facility is an example of how many health care workers will continue to work while ill despite the availability of sick leave . For instance, a study of 627 ambulatory care employees revealed that 83% of medical practitioners and about 64% of nurses, nurse s aides, allied health professionals, and administrative staff did not report taking sick leave when experiencing influenza - like illnesses37 . A systematic process that screens all employees for contagious illnesses at the start of their shift may limit the impact of presenteeism . The effectiveness of this intervention is unknown and requires further research, although active case finding is recommended by the cdc as an infection control measure for the h1n1 influenza pandemic38 . Policies also need to be in place that not only encourage but require staff to be excluded from work for a minimum amount of time after resolution of symptoms . The minimum amount of time required off may differ for various conditions, including 48 to 72 h for suspected viral gastroenteritis32 . The financial implications of mandatory exclusion policies for sick workers are likely to be considered a major barrier to widespread implementation . However, there is evidence that suggests that longer periods of worker exclusion may actually reduce the total number of sick days taken off within an institution due to an overall reduction in the number of ill staff members32 . Lastly, any policy that mandates strict back - to - work rules must also ensure adequate staffing and coverage of health care personnel to limit feelings of personal responsibility that encourage presenteeism39 . Despite the best efforts of education and mandatory exclusion rules, health care providers will likely continue to come to work if they feel that their absence would burden their colleagues or affect delivery of patient care8,24,25 . Furthermore, a policy that ensures adequate coverage may be cost - effective for health care institutions by mitigating the negative financial impact associated with large nosocomial outbreaks . There is evidence that decreased bed occupancy rates resulting from understaffed ward teams during a nosocomial norovirus outbreak result in greater costs to the institution than costs from infection control or diagnostic measures40 . Further research is needed to evaluate cost - effective ways to create some give in the system so that ill health care providers will believe their presence would be more of a risk than their absence . In the current era of frequent international travel and novel pandemic influenza virus outbreaks, vigilance is required to ensure that appropriate, common sense infection control procedures are in place, including enforcement of policies preventing health care staff from working while they are potentially infectious . These policies should include the availability of unrestricted paid sick leave, systematic processes for screening ill employees, and mandatory exclusion rules . A fundamental shift is necessary by health care organizations to view measures like unrestricted sick leave not solely as employee benefits, but rather as real investment opportunities that help protect patient safety.
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The latest definition of lpe includes 3 points: (i) ielt<1 minute; (ii) the inability to delay ejaculation; and (iii) negative personal consequences . Previous studies of twins and familial studies had proved the genetic predisposition in lpe [57]. A twins study in finnish men indicated that 0.30 of the etiology was a result of hereditary effect . However, twins and familial studies did not clarify which genetic variant might explain the genetic effects in lpe . To explore this problem, genetic association studies may be a possible approach . The susceptibility to lpe was considered to be a polygenic pattern, such as those genes linked to the metabolism of serotonin (5-ht) and those related to the expression of serotonin transporter (5-htt). Firstly, various selective serotonin reuptake inhibitors (ssris) were successfully used in the treatment of pe, and dapoxetine became the first approved drug for pe treatment . Secondly, animal experiments also demonstrated the role of 5-ht in ejaculation . Decreased duration of intravaginal ejaculation latency time (ielt) furthermore, snps located in genes encoding 5-ht receptors were also reported to play a role in ielt of patients with pe [1316]. Three common polymorphisms were described: an ins / del in the promoter, called 5-httlpr; a variable number of tandem repeat (vntr) in intron 2, called stin2; and a snp polymorphism in the noncoding 3-utr . In fact, 5-httlpr has been studied thoroughly, but the effect and function of stin2 vntr is not yet clear . It has been proved to include 10-repeat and 12-repeat alleles of a 16/17 bp element in all ethnicities, and the rare 9-repeat allele appears only in people of european or african descent . Stin2 vntr works as a transcriptional regulator, and the transcriptional activity is determined by the number of repeats, with the stin2.12 having the highest expression . Many studies had linked vntr with disorders such as depression, bipolar depression, and a predisposition to obsessive compulsive disorder . Although many studies have explored slc6a4 and lpe [2428], only 1 of them studied stin2 vntr, and there have been no such studies in chinese populations . Considering the important role of the serotonergic system in ejaculatory function, we aimed to determine whether stin2 vntr is related to lpe in china . Each participant was informed of the purpose of the study and signed the informed consent . The initial evaluation of each subject completed a detailed face - to - face interview by an andrologist, including a questionnaire and physical examination . Finally, 115 patients with lpe were recruited by referral from the andrology outpatient clinic . At the same time, subjects had to meet the following inclusion criteria: (i) heterosexual male patient aged 2060 years; (ii) han descent and speaking chinese; (iii) in a regular sexual relationship with 1 female partner for 6 months . None of the participants had received antidepressants or phosphodiesterase type 5 inhibitors before enrolling in the trial . Patients with a urinary infection or disorders of the nervous system were also excluded from the study . The study was approved by the ethics committee of the first affiliated hospital of anhui medical university (no.20150047). Genomic dna was extracted from peripheral blood using the puregene dna extraction kit (qiagen, hilden, germany) following the manufacturer s protocol . Oligonucleotide primers were as follows: forward 5-tggcgagatttgacttttctacc-3 and reverse 5-ctgagcttcatcaaggggaac-3 were used to amplify the stin2 vntr region . A 20-l mixture was ready for pcr amplification, including hotstartaq buffer, 3.0 mm mg, 0.3 mm dntp, 0.1m of primers, 1 u hotstartaq polymerase (qiagen, inc . ), and 1 l template dna . Pcr conditions were as follows: 15 min of initial denaturation at 95c; 11 cycles of 94c for 20 s and 620.5c per cycle for 40 s and 68c for 2 min; followed by 24 cycles of 20 s of denaturation at 94c, 56c for 40 s and 2 min of extension at 68c, and a final extension of 60 min at 60c . After digestion, the products were analyzed on a 3730 dna analyzer (abi, california). For the quantitative data, results are expressed as mean standard deviation (sd) and a 2-tailed t - test was used . After the normality and homoscedasticity test, analyses of variance (anova) using ielt as a variate were performed to test differences between different genotypes . Each participant was informed of the purpose of the study and signed the informed consent . The initial evaluation of each subject completed a detailed face - to - face interview by an andrologist, including a questionnaire and physical examination . Finally, 115 patients with lpe were recruited by referral from the andrology outpatient clinic . At the same time, subjects had to meet the following inclusion criteria: (i) heterosexual male patient aged 2060 years; (ii) han descent and speaking chinese; (iii) in a regular sexual relationship with 1 female partner for 6 months . None of the participants had received antidepressants or phosphodiesterase type 5 inhibitors before enrolling in the trial . Patients with a urinary infection or disorders of the nervous system were also excluded from the study . The study was approved by the ethics committee of the first affiliated hospital of anhui medical university (no.20150047). Genomic dna was extracted from peripheral blood using the puregene dna extraction kit (qiagen, hilden, germany) following the manufacturer s protocol . Oligonucleotide primers were as follows: forward 5-tggcgagatttgacttttctacc-3 and reverse 5-ctgagcttcatcaaggggaac-3 were used to amplify the stin2 vntr region . A 20-l mixture was ready for pcr amplification, including hotstartaq buffer, 3.0 mm mg, 0.3 mm dntp, 0.1m of primers, 1 u hotstartaq polymerase (qiagen, inc . ), and 1 l template dna . Pcr conditions were as follows: 15 min of initial denaturation at 95c; 11 cycles of 94c for 20 s and 620.5c per cycle for 40 s and 68c for 2 min; followed by 24 cycles of 20 s of denaturation at 94c, 56c for 40 s and 2 min of extension at 68c, and a final extension of 60 min at 60c . After digestion, the products were analyzed on a 3730 dna analyzer (abi, california). Results are expressed as mean standard deviation (sd) and a 2-tailed t - test was used . After the normality and homoscedasticity test, analyses of variance (anova) using ielt as a variate were performed to test differences between different genotypes . Among 216 subjects who met the inclusion criteria, only 1 patient was excluded from the present study because of sample coagulation . The patients and controls showed no significant difference in terms of any characteristic except age (35.37.8 vs. 31.78.1, p<0.001). However, because lpe is assumed be lifelong, the difference did not affect the comparability of the 2 groups . The most common allele was stin2.12 (patients: 82.5%, controls: 75.2%), and no significant difference was found between the 2 groups (p=0.182). The stin2.9/12 and stin2.9/9 genotypes were infrequent, so they were excluded from further analyses . The results showed a significantly higher frequency of stin2.12/12 genotype in lpe patients than in controls (p=0.026). Interestingly, the less common stin2.9 allele was present in our study, which was found in people of european or african descent in previous studies . Table 3 shows results of analyses under the 3 models: (i) dominant stin2 . 12 model (stin2.12/12+stin2.12/10 vs. stin2.10/10), (ii) dominant stin2.10 model (stin2.10/10+stin2.12/10 vs. stin2.12/12), and (iii) codominant model (stin2.12/12 vs. stin2.12/10 vs. stin2.10/10). To investigate the association between the 3 models and the risk of pe, binary logistic regression models were fitted . In the codominant model, we found a significantly lower frequency of stin2.12/10 in the lpe patients (23.0% vs. 35.7%, or=0.52, 95% ci=0.280.96), although the difference was not statistically significant (p=0.108). A statistically significant association of the dominant stin2.10 model of stin2 was observed . In this model, the combined frequency of stin2.10 carriers was significantly lower in patients with lpe when compared with controls (28.3% vs. 41.8%, or=0.55, 95% ci=0.310.97, p=0.040). The mean ielts were 22.6811.41 s, 33.629.13 s, and 33.179.26 s for stin2.12/12, stin2.12/10, and stin2.10/10, respectively . The results showed significant difference in patients with stin2.12/12 when compared to stin2.12/10 and stin2.10/10 genotypes (stin2.12/12 vs. stin2.12/10; p=0.001; stin2.12/12 vs. stin2.10/10; p=0.024). According to the stin2.10 dominant model, the ielt results of stin2.12/12 also showed a significant difference compared to stin2.12/10 + stin2.10/10 genotypes (p=0.001). The fold - increase of the mean ielt in the stin2.12/10 and stin2.10/10 genotypes compared to stin2.12/12 genotype was 1.48 . These data indicate that stin2.10 carriers on average had nearly a 50% longer intravaginal ejaculation time compared to stin2.12 homozygous in this study . Among 216 subjects who met the inclusion criteria, only 1 patient was excluded from the present study because of sample coagulation . The patients and controls showed no significant difference in terms of any characteristic except age (35.37.8 vs. 31.78.1, p<0.001). However, because lpe is assumed be lifelong, the difference did not affect the comparability of the 2 groups . The most common allele was stin2.12 (patients: 82.5%, controls: 75.2%), and no significant difference was found between the 2 groups (p=0.182). The stin2.9/12 and stin2.9/9 genotypes were infrequent, so they were excluded from further analyses . The results showed a significantly higher frequency of stin2.12/12 genotype in lpe patients than in controls (p=0.026). Interestingly, the less common stin2.9 allele was present in our study, which was found in people of european or african descent in previous studies . Table 3 shows results of analyses under the 3 models: (i) dominant stin2 . 12 model (stin2.12/12+stin2.12/10 vs. stin2.10/10), (ii) dominant stin2.10 model (stin2.10/10+stin2.12/10 vs. stin2.12/12), and (iii) codominant model (stin2.12/12 vs. stin2.12/10 vs. stin2.10/10). To investigate the association between the 3 models and the risk of pe, binary logistic regression models were fitted . In the codominant model, we found a significantly lower frequency of stin2.12/10 in the lpe patients (23.0% vs. 35.7%, or=0.52, 95% ci=0.280.96), although the difference was not statistically significant (p=0.108). A statistically significant association of the dominant stin2.10 model of stin2 was observed . In this model, the combined frequency of stin2.10 carriers was significantly lower in patients with lpe when compared with controls (28.3% vs. 41.8%, or=0.55, 95% ci=0.310.97, p=0.040). The mean ielts were 22.6811.41 s, 33.629.13 s, and 33.179.26 s for stin2.12/12, stin2.12/10, and stin2.10/10, respectively . The results showed significant difference in patients with stin2.12/12 when compared to stin2.12/10 and stin2.10/10 genotypes (stin2.12/12 vs. stin2.12/10; p=0.001; stin2.12/12 vs. stin2.10/10; p=0.024). According to the stin2.10 dominant model, the ielt results of stin2.12/12 also showed a significant difference compared to stin2.12/10 + stin2.10/10 genotypes (p=0.001). The fold - increase of the mean ielt in the stin2.12/10 and stin2.10/10 genotypes compared to stin2.12/12 genotype was 1.48 . These data indicate that stin2.10 carriers on average had nearly a 50% longer intravaginal ejaculation time compared to stin2.12 homozygous in this study . Recently, polymorphisms in slc6a4 have been increasingly associated with lpe, especially 5-httlpr polymorphism . Our study evaluated 114 patients and 101 controls for the relationship between stin2 vntr and lpe . The allelic frequency of the stin2.12 was 0.825 in patients and 0.752 in controls, which showed no significant difference (p=0.067). There was a significant difference in stin2.12/12 genotype, which was more common in the lpe group (p=0.026). However, our results contradicted a previous study by zuccarello et al ., which showed no difference between these 2 groups in genotypic and allelic frequency . More studies and meta - analyses of stin2 vntr polymorphisms in this field are needed . Our data demonstrate that stin2.12/10 and stin2.10/10 genotypes, which carry the lower transcriptional activity stin2.10 allele, were protective genotypes when compared with the stin2.12/12 genotype . A third major finding was that ielt in the patients group was associated with stin2 vntr . Therefore, the men carrying the higher expressing allele homozygote stin2.12/12 could have more functioning 5-htt, thus leading to lower 5-ht availability . On the one hand, gene frequencies of stin2 vntr vary significantly among different races and ethnicities, and is more common in japanese (0.997) than in african americans (0.713) and white americans (0.523). Our sample size of participants, as well as the relative homogeneity of demographic and clinical characteristics, make our study distinct . On the other hand, the function of stin2 vntr is still not clear . It may have a synergistic effect together with other polymorphisms on ejaculation, such as 5-httlpr ., only 1 study found that therapeutic effects were related to both stin2 vntr and 5-httlpr polymorphisms . The findings indicated stin2.12/12 genotype responded better to sertraline (p=0.001). We will plan to replicate the present study in this interesting field in the future . To the best of our knowledge, this is the first study to focus on the stin2 vntr polymorphism in chinese patients with lpe . Our present results indicate that stin2 vntr polymorphism is related to lpe . In summary, men with at least 1 copy of stin2.10 allele were at lower risk of pe than men with other variant at this locus . The current study shows that the stin2 vntr polymorphism is associated with ielts of lpe.
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Coronary artery disease due to atherosclerosis has emerged as a major social epidemic in india . It will soon emerge as the single largest disease accounting for nearly one - third of all deaths in india . According to estimates, it is projected that by the year 2015, coronary artery disease would cause around 2.95 million deaths, of which 14% of the mortality will be in population under 30 years of age and 31% deaths will occur in subjects below the age of 40 years . Atherosclerosis is a chronic degenerative condition of arteries responsible for significant cardiovascular morbidity and mortality worldwide . In the indian subcontinent, it is reported to be responsible for more than 25% of deaths . Atherosclerotic lesions start developing at an earlier age and are found to be in more advanced stages in indian population as compared to the patients in western countries . Atherosclerosis can lead to various complications like myocardial infarction (mi), stroke, embolization, ulceration, thrombosis, and aneurysm which cause considerable morbidity and mortality, thus affecting the lifespan and the quality of life of a large segment of population . Assessment of atherosclerotic lesions in living subjects is difficult and almost nonexistent due to its invasive nature and can be an expensive enterprise . Hence, autopsy - based study of coronary vessels and aorta, has emerged as an invaluable tool for studying these atherosclerotic lesions in deceased subjects . It will be a true representation of distribution and prevalence of atherosclerotic lesions present in the population if an autopsy study is conducted on deceased patients without any prior history of coronary artery disease and who expired due to noncardiac causes (as compared to the deaths caused by cardiac causes). With this hypothesis we conducted this study in 113 noncardiac death patients . This study was conducted from august 2012 to february 2013 at department of pathology, bj medical college, ahmedabad, gujarat . The deceased patients who underwent autopsy at our hospital and their prior / past medical history were recorded and their hearts were sent to our department for histopathological analysis . Written informed consent was taken from the relative / guardian of the deceased patient . The hearts of 250 successive autopsies were received in the department . Amongst these 113 deceased individuals had no prior history of cardiac disease, that is, deaths due to noncardiac causes . The hearts were fixed in 10% formalin, weighed, measured, and the three main coronary arteries were dissected out . A 5 cm section of the right coronary artery (rca) in the atrioventricular groove from its origin, a 5 cm segment of the left anterior descending artery (lada) distal to the origin of the circumflex artery, but including the region of origin of the circumflex branch and left coronary artery (lca) from its origin till the circumflex branch were excised and dissected out . The exposed artery was carefully examined for any thickening, yellow streaks, frank plaque, or calcification . After routine processing and paraffin embedding, 4 m sections were taken and stained with hematoxylin and eosin (h and e). Lipid core and fibrotic layer or multiple lipid cores and fibrotic lipid layers; mainly calcific or fibrotic . The degree of atherosclerosis was classified as unremarkable (grade 0), mild (grade 1 - 2), moderate (grade 3 - 4), and severe (grade 5 - 6). Lipid core and fibrotic layer or multiple lipid cores and fibrotic lipid layers; mainly calcific or fibrotic . The degree of atherosclerosis was classified as unremarkable (grade 0), mild (grade 1 - 2), moderate (grade 3 - 4), and severe (grade 5 - 6). During the study period from august 2012 to february 2013;250 consecutive autopsied hearts were submitted to the department of pathology, of which 113 deceased subjects had no prior history of coronary vessel disease and had a history of noncardiac cause of death . Out of these 113 subjects, 83 (73.45%) subjects had some histopathological evidence of coronary atherosclerosis . Amongst the 83 subjects with atherosclerosis, 68 (82%) were males and 15 (18%) were females [table 1]. The average heart weight for all 113 noncardiac death subjects in males and females was found to be 276.19 and 235.2 g, respectively . The average heart weight of 83 subjects found to have evidence of atherosclerosis in males and females was found to be 287.21 and 246 g, respectively . Age and sex distribution of atherosclerosis cases all the subjects were grouped into specific age groups based on the age at the time of death . The age and sex distribution of all 83 cases showing evidence of atherosclerosis the involvement of individual coronary arteries and severity of involvement is shown in table 2 . Degree / severity of atherosclerosis in the coronary vessels the 3 decade of life appears to be a watershed line in the pathogenesis of coronary vascular atherosclerosis, as we observed a steady increase in overall frequency, the number of coronary vessels involved, and severity of atherosclerosis from the 3 decade onwards [table 3]. Frequency and severity / degree of atherosclerosis in different coronary vessels before and after 3 decade of life amongst the 83 cases, all the three coronary arteries were severely involved (triple vessel disease) in 18 cases (22%). Double vessel and single vessel disease were seen in 10 (12%) and 11 (13%) cases, respectively [table 4]. Correlation of age with number of coronary vessels involved evidence of acute mi was found in nine (10.8%) cases, old healed scars were found in 11 (13.25%) cases, and evidence of congestion was found in 36 (43.37%) cases [table 5]. Morbidity and mortality due to coronary atherosclerosis in india has reached alarming proportions and these numbers are expected to maintain the upward trend in the next decade . Atherosclerosis is a commonly observed pathological finding in almost all ethnicities and societies worldwide, but with variable prevalence in different races . The onset of atherosclerosis starts early in life from childhood and gradually progresses through young adulthood to form the lesions that causes coronary heart disease later in life . In the present study, the overall incidence of atherosclerosis was found to be 73.45%, which is higher than what has been found in earlier studies by dhruva et al ., (23.3%); golshahi et al ., (28.9%); garg et al ., (46.4%); and yazdi et al ., (40%). Ischemic heart disease (ihd) due to coronary vascular disease is mainly caused due to atherosclerosis . The indian population is otherwise also vulnerable to coronary vascular disease and the disease also has an earlier onset in our population . In our study we found that there is a progressive steady increase in atherosclerosis of coronary vessels in individuals from the 3 decade of life onwards ., found that significant atherosclerotic lesions start developing from 2 decade of life and onwards . Tandon et al ., also reported that there was a progressive increase in the number and severity of atherosclerotic lesions from the 2 and 3 decade of life onwards . Dhruva et al ., also reported similar findings with increasing frequency of atherosclerosis from 3 decade onwards and with a peak of 76.6% in 6 decade, followed by a decline to 66.7% in the 8 decade . Garg et al ., also noted a progressive increase in atherosclerotic lesions from 3 decade onwards . In this modern globalized era, where human life style has become more and more complex and challenging . Various life stressors (anxiety, depression, etc .) Along with a sedentary lifestyle and lack of exercise and poor dietary habits like intake of junk food and increased use of refined and processed food items in place of whole grains and fresh fruits and vegetables can be important factors for earlier initiation of development and progressive increase in atherosclerotic lesions in this young indian population . Males have a relative preponderance of coronary heart disease as is evident from multitude of national and international studies conducted in the past . In the study conducted by garg et al ., they found coronary atherosclerotic lesions in 80.9% (93) males as compared to 19.1% (22) females . Bhargava and bhargava reported coronary atherosclerotic lesions were more prevalent in 74.8% males in comparison to 24.2% females in their study . Murthy et al ., studied 150 cases of coronary atherosclerotic lesions, out of which 123 (82%) were males and 27(18%) were females . Singh et al ., also reported coronary atherosclerotic lesions in 200 cases and found that these lesions were more frequently found in 85% males as compared to 15% in females . Padmavati and sandhu found that 66.5% males and 33.5% females were affected by coronary atherosclerotic lesions . In the present study too, we found a male (82%) preponderance of coronary atherosclerosis as compared to 18% in females . Moreover, there is greater indulgence of males in smoking and alcoholism as compared to females, which may possibly explain the male preponderance towards development of more severe and progressive atherosclerosis . In the 83 individuals with coronary atherosclerosis, the average weight of the heart was greater as compared to nonatherosclerotic cases this may be due to increased cardiac work load and compensatory hypertrophy . As atherosclerosis was found to be more in frequency and degree of severity in males as compared to females, it was also evident as the average hearts of males were heavier than those of age group - matched female patients . Our findings are comparable to the similar findings of dhruva et al ., and garg et al ., who too found that the average heart weight in males was higher as compared to females . Triple, double, and single vessel disease was found in 22, 12, and 13% of cases, respectively, with severe atherosclerotic lesions in our study . Dhruva et al ., found that single vessel was involved in 31%, while two and three vessel involvements was seen in 17 and 36% cases, respectively . In their study, garg et al ., reported that triple vessel involvement (44.4%) was the most common, followed by double and single vessel involvement seen in 42.2 and 13.3% cases, respectively . Incidence of coronary involvement in lada was 40%, rca 32%, and left circumflex artery 30% . This was in concordance with the data given by sudha et al ., who showed lada as the most common site for plaque (47%) and yazdi et al ., who showed lad as the most commonly involved artery (60%), followed by rca (50%) and left circumflex artery (42.5%) garg et al ., too found that lada (38.1%) was the commonest involved vessel followed by right coronary (35.1%) and left circumflex artery (34%). Acute mi was seen in 10.8% cases as compared to observations of 9.72, 6.5, and 3% acute mi cases observed by dhruva et al ., maru, and garg et al ., respectively . It is a bit surprising and unexpected finding that the percentage of acute mi in our study was slightly on the higher side as our subjects did not have any prior history of a coronary vascular event or history of cardiac cause of death . The study showed unexpectedly high prevalence of atherosclerosis in ahmedabad, india; especially in the relatively young population with no prior history of cardiovascular disease and where the diagnosis of coronary vascular disease is least suspected . However, there is a male preponderance of coronary atherosclerosis, but there is a progressive increase in the proportion of females who present with coronary atherosclerosis . The study of human atherosclerotic lesion is an extremely difficult task in a living subject and autopsy study is the best possible way to work on it . Though our study involved only a small number of cases, it highlights the early onset and increasing prevalence and severity of atherosclerotic lesions in indian population . It also calls for institution of screening programs and preventive and control measures against atherosclerosis from an early age.
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Corneal collagen crosslinking (cxl), using ultraviolet - a (uva) radiation and the photosensitizer riboflavin, was recently introduced for the treatment of progressive keratoconus.12 in several studies, the method has shown a positive effect on the biomechanical1345 and biochemical678 stability of the cornea . Thus, cxl has the potential to stop the progression of keratoconus, avoiding the need for penetrating keratoplasty.9 in regions of the world, where there is no access to corneal transplants, this simple and low priced procedure seems to offer the only accessible treatment for keratoconus patients at this time . Cxl requires a minimum central corneal thickness (cct) of 400 m to protect the endothelium from the cytotoxic irradiance.6 often, late diagnosed keratoconus corneas reach values considerably below this threshold . However, during cxl the stromal thickness can be influenced by the chemical composition of the applied riboflavin solution . Exposed to a physiologic aqueous medium the deepithelialized cornea shows the tendency to increase its volume (swelling pressure), which is mainly due to the concentration of stromal polysaccharides.10 recent in vitro studies in porcine corneas indicate that the osmolarity of the applied medium does not correlate with intraoperative corneal swelling.11 hence, the stroma can be hydrated or dehydrated during cxl, depending upon exposure to a riboflavin solution with a colloid osmotic pressure lower or higher than the physiological swelling pressure . The colloid osmotic pressure of the commonly applied cxl drop composition is mainly given by the polysaccharide dextran (20%), which is added to increase the viscosity of the solution . Dextran shows abundant hydrophilic hydroxyl groups and its solution leads to a marked intraoperative reduction of cct.12 logically, these drops are not adequate in controlling the 400 m threshold in patients with advanced stages of keratoconus . However, a number of alternative treatment protocols for thin corneas have been proposed.1314 one possibility is to apply a solution containing 0.5% of the polysaccharide methylhydroxypropylcellulose (mhpc). In comparison with dextran, mhpc is missing the water binding hydroxyl groups and its solution is known to swell the stroma quite effectively.15 it should be noted in this context, that cxl is expected to be less effective in artificially swollen corneas, due to the lower relative concentration of collagen in the hydrated stroma.1617 therefore, the stroma should be swollen as much as necessary, but as little as possible . In this pilot study, we introduced cxl in the eye clinic of bafoussam in the west region of cameroon . We applied variations of riboflavin solutions in order to deal with highly thinned out corneas . The major focus of this study was to determine the most effective treatment for each patient . In this every particular change of the riboflavin composition used is a result of our reaction to the given conditions . Cxl was performed in september 2011 and march 2012 in the eye clinic of bafoussam in the west region of cameroon . When we started treatment with the well - established european protocol, a study was not intended . However, during treatment, we found significant, unexpected differences between these african eyes compared with european eyes . Thus, we optimized our procedure step - by - step . Therefore, we did not apply for approval from the ethics committee and did not register the project with the institutional review board . Nevertheless, all the procedures were in accordance with the ethical standards of the declaration of helsinki . The diagnosis of keratoconus was based on corneal topography, ultrasound pachymetry and clinical signs, such as stromal thinning, vogt striae and apical stromal scars . Central corneal scars were not regarded as an exclusion criterion, as there was no keratoplasty treatment available for these patients . Drops containing mhpc were prepared following a protocol of the university hospital carl gustav carus in dresden . Drops with dextran and the drop composition without viscosity additive were produced according to protocols of the university medical center of the johannes gutenberg university in mainz . Solution 2 was produced in the pharmacy of the university medical center of the johannes gutenberg university in mainz . Initial weights for the production of 100 g riboflavin eye drops the treatment procedure was conducted under sterile conditions in the operating theater of the clinic . After instilling topical anesthesia (amethocaine 0.5%) and inserting a lid speculum, the central 9 mm of the corneal epithelium were abraded with a hockey stick spatula . The cct was measured by ultrasound pachymetry before and immediately after de - epithelialization and at least every 10 min during the 60 min of eye drop application . In order to detect the thinnest point, at least five repeat measurements at different positions on the central cornea the riboflavin solution was applied every 3 min for 30 min prior to irradiation to ensure sufficient saturation of the stroma . After confirming that the thinnest point of the corneal stroma was thicker than 400 m, uva - irradiation (3 mw / cm, 365 nm) the irradiation unit (uv-365) is an in - house production of the university medical center of the johannes gutenberg university in mainz . The irradiance was measured by the uva radiometer ilt 72 (international light technology, peabody, usa). The application of the riboflavin solution was continued every 3 min during irradiation . Using solution 6 the average age of patients was 17.9 4.7 years and ranged from 11 to 27 years . The treated eyes showed advanced stages of keratoconus with a mean maximum keratometry value of 61 4.3 d ranging from 53.1 to 68.8 d. the mean minimal corneal pachymetry including epithelium was 379 81 m ranging from 266 to 515 m [table 2]. Pre - operative and intra - operative findings figure 1 shows the mean cct as the percentage of the cct after de - epithelialization . During 60 min of application, solution 1 caused an intraoperative swelling of the corneal stroma to an average of 172 15% (n = 5). Application of solution 2 for 60 min resulted in a final cct value of 183 8% (n = 5). In the same time span, solution 3 led to a swelling up to 170% (n = 1). Solution 4 seemed to be proper for stabilizing the stromal volume, reaching a mean final cct of 99% after 45 min of application (n = 1). There was a marked thinning of final cct (80%) using solution 5 for 45 min (n = 1). Application of solution 6 for 50 min resulted in an increase in corneal thickness to a mean value of 150 13% after 50 min (n = 2). Mean central corneal thickness (cct) in percentages of the cct before treatment in 15 eyes after deepithelialization and application of various eye drop solutions . The graphs marked with * are measured after an initial swelling period with solution 6 solution 4 and 5 were applied in the context of our modified treatment procedure, which required an initial swelling of the corneal stroma considerably over 400 m and a subsequent stabilizing of the achieved cct value with an adapted dextran composition . The swelling of the corneal stroma was achieved by applying solution 6 and stabilization was achieved by applying solution 4 . Graph (b) shows the swelling and stabilizing period, using the modified protocol . Without exception, all known studies on cxl emphasize the challenge of keeping the stromal thickness of treated keratoconus corneas over the essential threshold of 400 m.1314 all the more surprising it seemed to us that we apparently had to deal with the contrary problem . Although we had to manage highly thinned out corneas, our main task consisted in keeping the intraoperative swelling within a limit . We treated five eyes with a riboflavin solution containing 0.5% mhpc (solution 1). This composition seemed to be particularly suitable for the treatment of thin african corneas, as a similar solution (riboflavin 0.2%/mhpc 0.5%/nacl 0.7%) caused an increase of mean final cct to 156% in caucasian corneas (n = 7).15 unexpectedly, this composition led to a considerably stronger intraoperative swelling in the treated african eyes, reaching a mean final cct of 172% . Although the post - abrasio values of the five treated eyes were below 400 m, they all swelled to values around 600 m during cxl [table 2]. Cxl is expected to lose effectiveness in artificially swollen corneas, due to the lower relative concentration of collagen in the hydrated stroma.1617 furthermore, the currently used treatment parameters are assumed to treat the anterior 250 m to 350 m.18 this suggests that corneas that are swollen too much are probably just cross - linked in the upper portion . Against this background our aim was to swell the corneal stroma as much as necessary and as little as possible . We increased the mhpc portion in the eye drops to 1.0% (soution 2) and 1.7% (solution 3). We tried to increase the colloid osmotic pressure of the solution, in order to counteract the swelling pressure of the cornea . With final ccts up to 781 m (solution 2) and 647 m (solution 3) in individual corneas furthermore, the increased mhpc portion reduced the practicality of the drops due to the rising viscosity . However, in the next step we tried to counteract the swelling of the stroma by allowing some loss to evaporation . The film of the applied mhpc solutions covers the cornea for approximately 32 min before it breaks up . This makes evaporation of corneal fluid into the air improbable.19 to allow evaporation, we treated two eyes using eye drops without colloid viscosity additive (solution 6). The break up time of these drops was expected to be close to 90 s.19 keeping a drop interval of 3 min, we assumed approximately 90 s after the break - up of the corneal film where the stroma could lose volume through evaporation . Supportingly, the dropping time before radiation was shortened to 20 min . In comparison with the drops based on mhpc, this solution showed a reduction of the intraoperative swelling, reaching a final cct of 150% . Nevertheless, the achieved absolute values of 658 m and 659 m were still too high to ensure effective treatment [table 2]. Considering these observations, it seemed that the intraoperative swelling could be modulated adequately by using a combination of swelling and stabilizing drop solutions . Swelling drops to bring the thin corneas over the necessary threshold of 400 m and stabilizing eye drops to keep the corneal thickness in a range approximately between 400 and 500 m which should ensure an effective treatment . Searching for a stabilizing riboflavin composition we referred to findings made in an in vitro study in porcine corneas . In this study, a negative correlation between the dextran concentration and the intraoperative swelling of the cornea was detected.11 accordingly, a drop composition with less than the currently used 20% reduces the intraoperative deswelling of the stroma probably due to the lower amount of hydrophilic hydroxyl groups in the solution and the decreased colloid osmotic pressure . First of all we swelled the stroma using eye drops without colloid viscosity additive (solution 6) clearly over the threshold of 400 m (542 m). Afterward we tried to stabilize the achieved value using a drop composition with 10% dextran (solution 5). In vitro, this drop composition caused a very slight increase of corneal thickness.11 however, in vivo the 10% dextran drops showed still a clear decrease of cct to 436 m during 45 min of application [table 2]. As these drops were obviously unsuitable for our intention we continued reducing the dextran concentration to 5% (solution 4). Once more we swelled the corneal stroma clearly over the 400 m threshold (517 m) using drops without viscosity additive (solution 6). In the following 45 min we applied the drops with 5% dextran . They only decreased the value to 510 m [table 2 and figure 2]. The question remains why the treated african corneas seemed to show a different swelling behavior in comparison with caucasian eyes . It is unlikely that climate such as temperature or humidity has an influence, as bafoussam (1500 m) is marked by a relatively mild climate . The assumption that the african origin itself might have an effect on the intraoperative swelling behavior is speculative . Apart from the awareness that african corneas are thinner than caucasians,2021 there are no other relevant differences described in the literature . However, we want to highlight one aspect, which does not necessarily have an influence on the swelling behavior, but which is remarkable nevertheless . Our treated patients in bafoussam had a mean age of 17.9 4.7 years, which on average, is 14 years younger than patients treated within the framework of a retrospective crosslinking study at the university medical center of the johannes gutenberg university in mainz, germany (n = 23).15 however, these young patients showed advanced stages of keratoconus . It is known that african american children have corneas that are on average 20 m thinner than those of white and hispanic children.22 however, this fact alone may not explain the early onset of the disease . Most likely this phenomenon is attributed to an increased eye rubbing, which was found in the medical history of all the treated patients . A multivariate study detected that the increased tendency of atopic patients to develop keratoconus can be attributed exclusively to eye rubbing.23 eye rubbing leads to repeated microtrauma and therefore to increased release of interleukin-1 . This modulates the apoptosis of keratocytes . Due to the genetically determined higher expression of interleukin-1-receptors, in the keratocytes of patients with keratoconus, an increased release of interleukin-1 could lead to a loss of keratocytes and stromal volume.24 there is no doubt that the use of individually adjusted swelling and stabilizing riboflavin compositions would bring the greatest benefit for each patient . To our findings, our recommended treatment protocol for thin african corneas provides an initial swelling of the corneal stroma to a value of 450 m, using a drop solution without viscosity additive . Certainly, these drops may also cause a slight stromal swelling or deswelling depending upon the treated patient, but under pachymetric control this method provides adequate protection of the endothelium and should ensure the greatest benefit for patients . 10 repeat measurements on different regions of the central cornea should be performed every 5 min . The increased number of repetitive measurements should be performed in order not to overlook the thinnest point . Our findings are not the result of an experimental set - up, but reflect our reactions to the unexpected intraoperative swelling behavior of the treated african corneas . As keratoconus is a rare disease we have not treated enough patients yet to consolidate the results achieved so far.
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The discovery and asymmetric synthesis of novel polyketides and their unnatural analogs fuel the development of new therapeutic agents . The structural complexity of this class of molecules has inspired and tested synthetic organic chemistry . Woodward pioneered the use of cyclic stereocontrol followed by ring - opening to reveal a single diastereomer of an acyclic target . For example, in the first synthesis of erythromycin a, a dithiadecalin template was employed to control relative stereochemistry of ensuing reactions; subsequent ring - opening provided a highly substituted acyclic polyketide . This general strategy has been applied successfully to the synthesis of many natural products . Ring - opening reactions of tetrahydrofurans and tetrahydropyrans have been developed; however, there are few examples that occur with formation of a new csp csp bond . Panek and co - workers have achieved diastereoselective ring - opening reactions of tetrahydropyrans with cyanide in the presence of a lewis acid (scheme 1a). The stereochemical course is consistent with a stereoablative reaction; minimization of a strain in a carbocation intermediate and attack of cyanide on the least hindered face provides the major diastereomer . A complementary approach to control of relative stereochemistry is via a stereospecific reaction, where stereochemical information is conserved throughout the transformation . Hoveyda and co - workers demonstrated that unsaturated cyclic ethers activated by pendant alcohols undergo stereospecific sn2 reactions with grignard reagents to yield enantioenriched acyclic products (scheme 1b). We sought to expand stereospecific ring - opening reactions to include saturated cyclic ethers that are not activated by ring strain . We envisioned stereospecific nickel - catalyzed ring - opening reactions of cyclic ethers, based on our enantiospecific kumada - type cross - coupling of ethers (scheme 1c). We anticipated that cross - coupling would proceed with inversion at the electrophilic carbon . Therefore, by appropriate choice of diastereomer of starting material 5, either the syn or anti diastereomer of 6 could be obtained selectively . This work would harness diastereoselective synthesis of tetrahydrofurans and tetrahydropyrans to provide complex acyclic fragments . The products would contain a free alcohol that could be further utilized in synthetic sequences . This method would provide stereospecific incorporation of a benzylic methyl substituent, a common motif in medicinal agents . In addition, strategic use of extended alkyl or aryl grignard reagents would allow for the generation of a wide range of unnatural polyketide analogs for biological testing . In this manuscript, we report the stereospecific kumada - type cross - coupling of tetrahydrofurans and tetrahydropyrans with a range of grignard reagents . We also report stereospecific negishi - type cross - coupling reactions of benzylic lactones with dimethyl zinc to provide enantioenriched carboxylic acids . To establish nickel - catalyzed ring - opening of cyclic ethers and determine the stereospecificity of the reaction, we designed model substrate (r)-7 based on our prior experience developing kumada - type cross - coupling reactions of benzylic ethers . We chose to first examine coupling with methyl grignard reagent, as incorporation of magic naphthyl - substituted tetrahydropyran (r)-7 is straightforward to prepare in high enantiomeric excess (ee) utilizing the corey bakshi we were pleased to see that in the presence of a nickel catalyst and grignard reagent, (r)-7 provided acyclic alcohol (s)-8 with cross - coupling at the benzylic center (scheme 2). The reaction was highly enantiospecific, providing the product in 96% ee and> 99% enantiospecificity (es). No reaction occurs in the presence of grignard reagent in the absence of nickel catalyst . We envisioned that the most powerful application of this method would be in ring - opening reactions of heterocycles containing multiple stereogenic centers . To test our hypothesis that the nickel - catalyzed ring opening would occur with inversion at the electrophilic carbon, irrespective of the presence of other stereogenic centers, we examined both diastereomers of substituted tetrahydrofuran 9 . In the presence of ni(cod)2 and dpephos each diastereomer underwent cross - coupling with clean inversion at the site of oxidative addition (scheme 3). Tetrahydrofuran trans-9 (dr> 20:1) afforded acyclic anti-10 in 93% and a dr of> 20:1 . The other diastereomer, cis-9 (dr> 20:1), afforded syn-10 in 93% yield and> the relative configuration of tetrahydrofuran cis-9 was determined by x - ray crystallographic analysis . The relative configuration of both diastereomers of acyclic 10 was assigned based on analysis of chemical shifts in the h nmr spectra, based on the pioneering strategy of kishi for assignment of relative configuration of acyclic polyketide fragments using the breit model for 1,3-deoxypropionates . To determine whether or not there is a match / mismatch effect in reactions employing chiral catalysts, we examined ring - opening of both diastereomers of tetrahydrofuran 11 with each enantiomer of binap (table 1). If the reaction proceeds strictly with inversion, regardless of the catalyst chirality, then both enantiomers of binap would provide similar results in the ring - opening reactions . However, if the chiral catalyst influences the stereochemical outcome of the cross - coupling reaction, then one enantiomer of binap should provide diminished or inverted diastereoselectivity . In reactions of tetrahydrofuran cis-11 both enantiomers of binap afforded acyclic syn-12 in similar yield and 20:1 dr (entries 1 and 2). Similarly, use of either enantiomer of binap in reactions of trans-11 provided anti-12 in good yield and 20:1 dr (entries 3 and 4). Therefore, we conclude that there is no match / mismatch between the chirality of the catalyst and substrate . All reactions proceed strictly with inversion without influence by the chirality of the catalyst . These results are consistent with our previous observations of robust substrate control in stereospecific kumada and negishi coupling reactions . We next examined the application of the methodology to a series of substituted tetrahydrofurans with a broad array of substituent patterns and stereochemical relationships found in polyketides . Our starting materials were 2-aryltetrahydrofurans, a motif at the core of natural products such as the lignans sesaminone and pinoresinol . As such, there are outstanding methods for diastereoselective synthesis of highly substituted 2-aryltetrahydrofurans . Furthermore, development of methods for their direct derivatization could have application in natural product editing . We prepared substrates using the general strategy outlined in scheme 4, employing lewis - acid catalyzed cyclization of the requisite diols (see si for full details). This synthesis typically provides access to both diastereomers, important for interrogation of the influence of additional stereogenic centers on the stereochemical course of the cross - coupling reactions . The relative configuration of starting materials and products could be assigned using well established methods typically employed for polyketides, including noe experiments, analysis of h nmr spectra, and x - ray crystallography . We continued our studies with 2,4-disubstituted tetrahydrofurans, as ring - opening provides the 1,3-dimethyldeoxypropionate fragment (e.g., 9 to 10, scheme 3). To determine the stereochemical course of the reaction, we examined reactions of both diastereomers of tetrahydrofuran 11 (table 2, entries 1 and 2). Under kumada - type coupling conditions, tetrahydrofuran trans-11 (dr> 20:1) afforded acyclic anti-12 in 82% and> 20:1 dr (entry 1). The diastereomer, cis-11, (dr 9:1) gave syn-12 in 86% and 9:1 dr (entry 2). Rigorous assignment of the relative configuration of the starting materials and products demonstrates that the kumada - type couplings proceeded with inversion . We next examined introduction of a substituent at the 5-position, to determine whether the method would be amenable to synthesis of secondary alcohols . Both diastereomers of 2,5-disubstituted tetrahydrofuran 13 were synthesized by the stetter reaction, reduction of the ketones and diol cyclization . Tetrahydrofuran cis-13 (dr 9:1) underwent cross - coupling in 82% yield with inversion to yield syn-14 (dr 9:1, entry 3). The diastereomer trans-13 (dr 8:1), afforded anti-14 with high stereospecificity and in a slightly lower yield of 61% (entry 4). This lower yield is presumably due to steric interactions with the pseudo axial c5 methyl that would be present in the trans diastereomer but not in the cis diastereomer . Vicinal methyl - bearing stereogenic centers are motifs in natural products such as kalkitoxin and nordihydroguaiaretic acid . Starting materials trans-15 and trans-17 were prepared in 9:1 dr using the nozaki hiyama kishi reaction as a key step . Using dpephos as the ligand, trans-15 afforded syn-16 in 65% yield and 9:1 dr (entry 5). In addition to benzofuran - substituted tetrahydrofuran 9, benzothiophene - substituted trans-17 (dr 9:1) afforded syn-18 in good yield and 9:1 dr (entry 6). While coupling reactions of both 15 and 17 proceed with inversion, more modest yields are likely an impact of steric crowding near the site of oxidative addition . We examined 4-methoxytetrahydrofurans for synthesis of a 1,3-disubstituted ether unit as found in fk-506 and geldanamycin . We found that methoxy - substituted tetrahydrofurans trans-19 and cis-19 underwent the reaction to afford good yields and excellent transfer of stereochemical information (entries 7 and 8). We attribute slightly diminished yields with both diastereomers of 19 to formation of an allylic ether via -hydride elimination, which further reacted to a mixture of products under the reaction conditions . Reaction performed using dpephos (10 mol%) instead of rac - binap . Nap = 2-naphthyl . To further expand the scope and utility of this method to include synthesis of 5-substituted alcohols, we next turned our attention to tetrahydropyrans . We chose to examine cis-()-2,4-disubstituted tetrahydropyrans, subunits of the calyxin family of natural products . There are several elegant methods for the diastereoselective synthesis of highly substituted tetrahydropyrans . For example, these tetrahydropyrans are easily accessed by diastereoselective prins cyclization reactions, as outlined in scheme 5a . Clay - mediated prins cyclization of 2-napthaldehyde with 4-buten-1-ol in benzene provided cis-()-21 in a single step with high diastereoselectivity and reasonable yield . (a) montmorillonite k10 (1.1 equiv), meoh, c6h6, reflux, 18 h; (b) p - tsa (1.0 equiv), mgbr2 (1.1 equiv), ch2cl2, rt, 18 h; (c) ni(cod)2 (10 mol%), bathophenanthroline (bphen) (20 mol%), arb(oh)2 (1.2 equiv), kotbu (1.6 equiv), s - buoh, 60 c, 24 h. we developed an alternative two - step diastereoselective strategy to prepare tetrahydropyrans containing a broad range of aryl substituents at the c4 position (scheme 5b). 4-bromotetrahydropyran 22 is easily synthesized as a 2:1 mixture of diastereomers under mild conditions via a mgbr2 and p - tsoh - promoted prins cyclization . To further derivatize 22, we employed a nickel - catalyzed suzuki - type cross - coupling reaction . Based on the seminal work of fu, we hypothesized that the coupling would be stereoconvergent and afford the more stable diastereomer, cis-23 . Indeed, cross - coupling of 22 with a range of commercially available aryl boronic acids afforded a wide variety of 4-aryltetrahydropyrans in high diastereoselectivity . These results are consistent with a stereoablative cross - coupling reaction that proceeds through a radical intermediate, with a strong preference for formation of the thermodynamic product . The relative configuration of these cis-2,4-diaryl tetrahydropyrans was assigned by x - ray crystallographic analysis and noe nmr experiments (see si for details). As with the tetrahydrofuran substrates, we examined the transfer of stereochemical information in the cross - coupling reaction by comparing the diastereomeric ratios of the starting materials to those of the acyclic products . We observed that employing a catalyst loading of 15 mol% resulted in good to excellent yields with high diastereomeric ratios (table 3). Tetrahydropyran 21 (dr> 20:1) afforded syn-24 in 84% yield and> 20:1 dr (entry 1) indicating the complete transfer of stereochemical information in the cross - coupling . We found that both electron - rich and electron - poor aryl substituents at the c4 position of the tetrahydropyran are well tolerated in the reaction (entries 2 and 3). To further challenge the tetrahydropyran ring - opening for example, the cross - coupling of tetrahydropyran cis-29 proceeded in 81% yield and> 1,4-benzodioxanes are present in a range of pharmaceutical agents such as piperoxan and idazoxan . We were also pleased to see that 3-furan - substituted tetrahydropyran cis-31 was well tolerated in the reaction . Product syn-32 was formed in high yield and dr and contains a furan substituent that can be readily derivatized by oxidation or cycloaddition reactions (entry 5). Reaction performed using ni(acac)2 (10 mol%) and dpephos (10 mol%) instead of rac - binap ., we examined kumada coupling of 2,4,6-trisubstituted tetrahydropyran cis-33 . Subjecting cis-33 to the reaction conditions afforded the secondary alcohol syn-34, containing three stereogenic centers, as a single diastereomer and with good yield (entry 6). This strategy provides a modular three - step synthesis of polyketide analogs where substituents in the c2, c4, and c6 positions can be easily altered by the use of commercially available aldehydes, arylboronic acids, and homoallylic alcohols, respectively . We set out to determine the compatibility of the reaction conditions with silylethers, common protecting groups . 4-hydroxytetrahydropyrans are straightforward to prepare in high diastereoselectivity by prins cyclization employing trifluoroacetic acid . Using dpephos, we found that benzofuran- and benzothiophene - substituted tetrahydropyrans 35 and 37 afforded the acyclic products in good yields and> 20:1 dr (entries 7 and 8). To further challenge the ring - opening reaction, we employed a substrate activated by a simple aromatic substituent . 3-furan - substituted tetrahydropyran cis-39 formed syn-40 in high yield and> 20:1 dr (entry 9). The ability of a synthetic method to easily provide access to analogs with a range of substituent patterns is critical for the discovery of new therapeutics as well as for conducting structure activity relationship (sar) studies . We next wanted to examine the scope with respect to the transmetallating agent, which would provide access to a variety of benzylic substituents in the acyclic products . Our laboratory has recently demonstrated that ni(dppe)cl2 is a broadly applicable catalyst for cross - coupling of alkyl and aryl grignard reagents with benzylic ethers . To examine the generality of conditions for a range of grignard reagents, we applied this catalyst system to the cross - coupling reactions of a representative series of tetrahydropyrans and tetrahydrofurans (table 4). Cross - coupling with tetrahydrofuran 19 afforded the desired product in 75% yield and high dr (entry 1). 3-phenylpropylmagnesium bromide reacted smoothly with tetrahydropyran 37 to form alcohol 42 in good yield and high dr (entry 2). Phenylmagnesium bromide underwent successful cross - coupling with tetrahydrofuran 9 and tetrahydropyran 21 (entries 3 and 4). In order to confirm that the reaction proceeded with inversion at the benzylic position, both trans-19 and cis-19 were prepared and subjected to the kumada couplings with phenylmagnesium bromide . As with methylmagnesium bromide, the products were afforded in good yield and in high dr (entries 5 and 6). The relative configurations of anti-45 and syn-45 were assigned by the preparation of crystalline derivatives that were subjected to x - ray crystallographic analysis (see si for details). Products 46 and 47 were formed in high yield and dr (entries 7 and 8, respectively). We have previously demonstrated that diarylalkanes containing a thiophene moiety provide lead compounds with selective anti - breast cancer activity . We therefore also examined our methodology with 2-thiophenylmagnesium bromide as the nucleophile . With both 2,4-disubstituted tetrahydropyran 21 and tetrahydrofuran 11 the desired product was afforded in excellent yield and dr (entries 9 and 10). We hypothesized that a similar cross - coupling could be applied to benzylic lactones, based on our recently reported negishi - type nickel - catalyzed cross - coupling of benzylic esters . Sawama and co - workers have recently demonstrated that aryl substituted lactones undergo lewis acid - catalyzed ring - opening with allylsilane . However, to the best of our knowledge, stereoselective ring - opening of lactones with carbon - based nucleophiles has not been reported . As a further benefit, the resulting enantioenriched product would contain a benzylic stereocenter with a distal carboxylic acid . As with the alcohols obtained from the kumada - type opening of cyclic ethers, this carboxylic acid affords a convenient synthetic handle that can be used for further derivatization . We prepared enantioenriched lactones for cross - coupling by cbs reduction of the benzylic ketones and cyclization of the corresponding 1,5-diols . The absolute configuration of the lactones were assigned as r by the accepted cbs model of selectivity of the intermediate alcohols (see si for details). After examining a series of bidentate ligands and nickel precatalysts, we determined that ni(acac)2 and xantphos afforded the highest yield of cross - coupled carboxylic acid . Commercially available lactone 50 afforded the cross - coupled product with excellent es (table 5, entry 1), as did benzofuran - substituted lactone 52 and indole - substituted lactone 54 (entries 2 and 3, respectively). Furan - substituted -valerolactones such as 56 are found in natural products such as ricciocarpin a and salvinorin b; methods for their ring - opening would provide a strategy for synthesis of analogs for biological testing . We anticipated that lactone 56 would undergo straightforward nickel - catalyzed negishi - type cross - coupling . Our laboratory has observed a strong dependence of the rate of cross - coupling on the identity of the aryl substituent . We hypothesize that arenes possessing lower aromatic stabilization energy are better ligands for the nickel catalyst and stabilize the transition state for oxidative addition . Benzylic ethers and esters activated by extended aromatic rings such as naphthalene and benzofuran are sufficiently reactive, as are those activated by furan . Furthermore, the incorporation of the furan moiety affords a product with two functional group handles: the carboxylic acid and the furan itself . Therefore, we evaluated a 3-furan - substituted lactone and found that (r)-56 underwent the cross - coupling with 84% yield and> 99% es (table 5, entry 4). To take advantage of the furan s utility for further manipulations, we derivatized product (s)-57 by a diels alder reaction (scheme 6). The cycloaddition furnished the enantioenriched bicyclic acid 58 in 64% yield as a 1:1 mixture of diastereomers . Based on woodward s analysis of the thermodynamic product of the reaction, the diels alder reaction is anticipated to be highly exo selective . Yield determined by h nmr based on comparison to phtms as internal standard . Dyslipidemia, a serum lipoprotein level disorder, is implicated in cardiovascular diseases and is often treated with niacin . Anti - dyslipidemia agent 61 was disclosed as part of a campaign for discovery of niacin receptor agonists with reduced side effects . Amide 61 was previously synthesized in seven steps and used chiral chromatography to separate the enantiomers . (a) ni(acac)2 (10 mol%), xantphos (20 mol%), znme2 (3.0 equiv), phme, rt, 24 h; (b) (i) (cocl)2 (1.3 equiv), c6h6, rt, 2 h; (ii) anthranilic acid (1.1 equiv), c6h6, rt, 3 h. we applied our methodology to the asymmetric synthesis of niacin receptor agonist 61 from commercially available lactone (r)-59 (scheme 7). Utilizing our optimized cross - coupling conditions, carboxylic acid (s)-60 was afforded in 76% yield with> 99% es . A subsequent amide coupling therefore, using our method either enantiomer of anti - dyslipidemia agent 61 can be prepared in two steps and 57% overall yield from commercially available starting material . In summary, we have developed the nickel - catalyzed, stereospecific ring - opening cross - coupling reactions of aryl - substituted tetrahydrofurans, tetrahydropyrans, and lactones . Through judicious choice of starting materials, cyclic ether intermediates have been utilized to set the desired relative stereochemical relationships and allow for the selective synthesis of syn- and anti- deoxypropionate subunits . We have demonstrated the high stereospecificity of the reaction, where the dr of the product matches the dr of the starting o - heterocycles . The negishi - type cross - coupling of benzylic lactones has allowed for the enantiospecific synthesis of enantioenriched carboxylic acids, which can be further derivatized . Using this methodology, we report the two - step, enantiospecific synthesis of an anti - dyslipidemia agent with easy access to either enantiomer . We are currently investigating the application of these methods toward the implementation of natural product editing to generate a library of unnatural polyketides for sar studies.
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Chiari i malformation is a heterogeneous entity characterized by impaired cerebral spinal fluid (csf) circulation at the level of the foramen magnum due to cerebellar tonsillar ectopia . Less common presentations include motor deficiencies, lower cranial nerve palsies, sensory dysfunctions, nystagmus, vertigo, ataxia, dysphagia, apnea, and snoring . Several cases of associated syncope have been reported as a presenting symptom caused by craniovertebral junction anatomic abnormalities.1 2 3 4 5 6 7 8 advanced lesions associated with chiari malformations and assimilation of the atlas may compress the medulla upon the clivus or cause temporary vascular occlusion resulting in brainstem ischemia and abnormalities in heart rate and respiration.9 10 we here present a patient harboring both a chiari i malformation and atlanto - occipital assimilation contributing to cough syncope, orthopnea, and apnea . Chiari i malformation is a heterogeneous entity characterized by impaired cerebral spinal fluid (csf) circulation at the level of the foramen magnum due to cerebellar tonsillar ectopia . Less common presentations include motor deficiencies, lower cranial nerve palsies, sensory dysfunctions, nystagmus, vertigo, ataxia, dysphagia, apnea, and snoring . Several cases of associated syncope have been reported as a presenting symptom caused by craniovertebral junction anatomic abnormalities.1 2 3 4 5 6 7 8 advanced lesions associated with chiari malformations and assimilation of the atlas may compress the medulla upon the clivus or cause temporary vascular occlusion resulting in brainstem ischemia and abnormalities in heart rate and respiration.9 10 we here present a patient harboring both a chiari i malformation and atlanto - occipital assimilation contributing to cough syncope, orthopnea, and apnea . A 54-year - old man with history of sleep apnea presented to our neurosurgical clinic with complaints of progressive orthopnea and multiple episodes of cough syncope . The patient preferred to sleep upright to approximately 45 degrees for the past year; otherwise, he would wake up gasping for air . He was initially diagnosed with an anxiety disorder and was started on lorazepam, which failed to provide relief . Later, he was given the diagnosis of sleep apnea, which was unsuccessfully treated with continuous positive airway pressure (cpap). These episodes of orthopnea progressed to the point where he would become apneic while awake in a horizontal position . His syncopal episodes were significant in that they had resulted in motor vehicle accidents on two separate occasions . Initial syncopal workup at another institution included an extensive cardiopulmonary investigation that suggested a possible cardiac dysrhythmia . Subsequently, he underwent cardiac radiofrequency ablation . However, his syncopal episodes associated with coughing increased in frequency . Furthermore, he reported 1 years of progressive imbalance, subjective quadriparesis, dysphagia, headache, and hoarseness . His physical exam was significant for bilateral nystagmus on far lateral gaze, downbeat nystagmus on downward gaze, hoarse voice, and weak palatal elevation . The patient did have good strength throughout all muscle groups but was diffusely hyper - reflexic, though no pathologic reflexes could be elicited . The patient's magnetic resonance imaging (mri) demonstrated a chiari i malformation with associated assimilation of the atlas with the occipital bone, reducing the size of the foramen magnum (fig . T2- weighted sagittal (a) and t1-weighted contrasted coronal (b) magnetic resonance imaging (mri) demonstrates a chiari i malformation and assimilated atlas . The dimensions of the foramen magnum here measure 25 mm on anterior - posterior diameter and 35 mm on transverse diameter . Cervical x - rays in neutral (a), flexion (b), and extension (c) positions again demonstrate evidence of an atlanto - occipital assimilation . Our recommendation, in light of the patient's progressive and profound neurological symptoms, was for surgical decompression the following morning after his evaluation in the clinic . The patient underwent a chiari decompression, consisting of a suboccipital craniotomy, cervical level 2 laminectomy, and duraplasty with suturable duragen (integra, plainsboro, new jersey, united states) and duraseal (covidien, mansfield, massachusetts, united states). However, his other neurological symptoms including orthopnea, strength, and hoarseness had significantly improved . On postoperative day 3, the patient was taken back to the operating room for a wound revision for a csf leak, which was repaired, proven by its resistance to a valsalva maneuver . The rest of his stay was uncomplicated and he was discharged home on postoperative day 7 . Between weeks 2 and 4 after discharge, the patient progressively developed headaches similar in character to his preoperative condition . An mri was done at that time, which revealed a large pseudomeningocele and likely continued compression at the cervical 3 (c3) level (fig ., we had recommended extending the decompression via a c3 laminectomy, csf leak repair, and placement of a lumbar drain . The patient's symptoms had resolved via the combined interventions, and he was discharged home on postoperative day 4 . One month postoperative sagittal (a) and axial (b) t2-weighted magnetic resonance imaging (mri) demonstrating large pseudomeningocele at the site of the suboccipital craniectomy and upper cervical laminectomies extending into the posterior neck soft tissues, measuring approximately 7.7 6 cm and causing mass effect on the inferiorly migrated cerebellar tonsils . On 1-year follow - up, the prevalence of a chiari i malformation is estimated to be in the range of 1 per 1,000 to 1 per 5,000 individuals.11 the extent of atlanto - occipital assimilation varies from partial to total, which may account for the broad reported incidence from 0.08% to 3.6%.12 13 chiari i malformation readily occurs in the presence of developmental bony anomalies.13 14 in a report of 364 cases of symptomatic chiari i malformations, anomaly of the occipital bone including occipitalization of the atlas, condylar hypoplasia, and other atypia of the occipital bone itself did not exceed 5.2%.6 conversely, some authors suggest that chiari malformation exists in approximately 50% of patients with atlanto - occipital assimilation, generating possible csf flow disorders.15 the occipital bone that forms the foramen magnum and vertebra originate in sclerotomes at the ventromedial portion of somites separated from the para - axial mesoderm . These structures are formed through complex differentiation processes that occur from gestational weeks 4 to 12.16 maldevelopment may lead to craniovertebral junction abnormalities with varying degrees of superimposed bony anomalies,12 13 17 18 19 such as assimilation of the atlas, which is defined as a failure of segmentation between the fourth occipital sclerotome and the first spinal sclerotome.20 21 as a result, the volume of the posterior cranial fossa and foramen magnum is often reduced, which potentially produces distortion and compression of neural structures.22 23 several morphometric analyses of the foramen magnum in dry skulls report an average anterior - posterior (ap) length to be 33.3 to 35.33 mm and the average transverse diameter to be 27.9 to 29.67 mm.24 25 26 27 in our case study, the patient's ap length was 25 mm (approximately 25% reduction) and his transverse diameter was 29 mm . In our case report, the patient unfortunately required two further surgeries to correct a likely continued csf obstruction that was inadequately initially addressed . After both the initial suboccipital decompression / c2 laminectomy and the subsequent wound revision to repair the csf leak, we were able to achieve a watertight closure proven by valsalva maneuver . Therefore, we hypothesize that the profound narrowing associated with this rare anatomical combination of chiari i malformation and atlanto- occipital assimilation may require a more extensive evaluation in regards to extent of decompression . Data provided by such studies as preoperative cine mri (csf pulsatile flow study) at the foramen magnum or intraoperative ultrasound to assess adequacy of csf flow from the fourth ventricle may have prevented this postoperative complication . Other anatomical considerations in managing patients with such bony anomalies include cognizance that the vertebral arteries may be malformed or pass abnormally and that the craniovertebral junction is a site inherently subject to instability.13 therefore, in the presence of bony anomalies, it is important that management is determined on morphologic examination supplemented with a dynamic image - based evaluation of instability involving anterior and posterior flexion or horizontal rotation of the head.28 classically, patients with chiari malformation present with pain, weakness, sensory loss, dizziness, gait disturbances, or cranial nerve palsies . In patients with chiari malformations, systematic studies have shown a prevalence of sleep respiratory disturbances between 59 and 70%.29 30 however, syncope is a relatively rare symptom and may occur with or without premonitory symptoms . Although the exact cause of chiari i malformation - related syncope is not well understood, various pathophysiological mechanisms have been proposed that may occur with transient increases in intracranial pressure with valsalva maneuvers, such as the following: vertebrobasilar artery compression, compression of the midbrain ascending reticular system, and compression of cardiorespiratory centers or their efferent and afferent autonomic pathways.3 4 6 31 32 several isolated case reports of severe respiratory dysfunctions improved by decompressive surgery have been described.33 34 however, there are a few more systematic studies regarding the effects of surgery on sleep respiratory dysfunction in patients with craniovertebrobasilar junction malformations . One study evaluated 16 patients and found sleep apnea in 12 of them, with 48% of cases being central apnea.35 eight patients were surgically treated, and six underwent postoperative polysomnography, which indicated decreases in the patients' apnea index (defined as total number of apnea and hypopnea events divided by total sleep time) from 23.5 7.9 to 9.8 6.6 (p = 0.1) and decreases in their central apnea index (cai) (defined as number of sleep apneas divided by total sleep time) from 14.9 5.5 to 1.3 0.6 (p = 0.03). A similar study prospectively evaluated 25 patients with chiari malformations who were then subjected to full - night polysomnography.36 seventeen patients were diagnosed with sleep apnea, seven of which were found to have central apnea . After posterior fossa decompressive surgery, postoperative polysomnography demonstrated a decrease in apnea index from 26.68 to 12.98 (p = 0.06) and mean cai from 13.81 to 1.68 (p = 0.01). In patients with cervicomedullary anatomic abnormalities due to chiari deformities, ireland et al32 evaluated both preoperative and postoperative postural changes in heart rate, heart rate variability, and blood pressure in nine patients with chiari malformations and cough syncope . Their data suggested that patients with cervicomedullary anatomic abnormalities that may have abnormal autonomic control of heart rate return to a normal pattern after surgical palliation in conjunction with resolution of clinical symptoms . With decompression of the cervicomedullary junction, the csf pathways are reformed, eliminating the pressure gradient between the cranium and spine and further impaction of the cerebellar tonsils . Although chiari i malformation related syncope is rare, it should be considered amongst the differential diagnosis in working up a syncopal episode . Concomitant with atlanto - occipital assimilation, the reduction in size of the foramen magnum may result in profound respiratory abnormalities that may require a more extensive decompression.
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Non - alcoholic fatty liver disease (nafld) is the most common chronic liver disease in north america and has emerged as a serious public health burden . The clinical - histological phenotype of nafld extends from non - alcoholic fatty liver (nafl) to nash . The estimated worldwide prevalences of nafld and nash range from 6.3%-33% and 3%-5%, respectively . Whereas nafl can progress to cirrhosis in 2% to 3%, nash has an increased risk for the progression to cirrhosis at 15% to 20% and predisposes patients to the development of hepatocellular carcinoma and increased mortality . Although extensive research in understanding the disease pathogenesis and numerous clinical trials aimed at halting disease progression have been performed in the last two decades, optimal therapy is still lacking . The aims of this article are to provide a short overview of important mechanisms and genetic factors influencing nafld disease progression and to present strategies in the diagnostic and therapeutic management of nash . Dysfunctional adipocytes secrete cytokines and chemokines, and this perpetuating inflammatory cycle induces a state of insulin resistance, resulting in failure to suppress lipolysis in the adipocytes . This leads to release of free fatty acids (ffas) to the circulation and is followed by uptake of these ffas to the liver . A carbohydrate - rich diet and hyperinsulinemia activate carbohydrate - related element - binding protein (chrebp) and sterol regulatory element - binding protein-1 (srebp-1), respectively, and promote hepatic de novo lipogenesis . The imbalance between triglyceride acquisition and removal results in excess triglycerides stored as lipid droplets . Key players include lipotoxicity, oxidative stress, endoplasmic reticulum (er) stress, activation of the innate immune activity, and cytokine - mediated cellular damage . It has been demonstrated that triglyceride synthesis is not harmful but protective against fatty acid - induced lipotoxicity . It is known that ffas can directly activate inflammatory pathways, er stress, and the innate immune system via toll - like receptors . The presence of biologically active lipid molecules such as free cholesterol, diacylglycerol, lysophosphatidylcholine, and ceramides can affect lipogenesis, insulin signaling, and cellular injury and contribute to nafld phenotype and disease progression [1012]. Once the capacity of the liver to store triglycerides is overwhelmed, there is increased compensation by the mitochondria and peroxisomes to oxidize fatty acids . The oxidative capacity of these organelles becomes impaired and can lead to overproduction of reactive oxygen species . The consequent cytokine production and lipid peroxidation have the potential to induce apoptosis, inflammation, and liver fibrosis . In response to er stress, the unfolded protein response moreover, the gut microbiome has been more recognized to play an important role in the pathogenesis of nash . Changes in the microbiota can alter intestinal permeability and promote translocation of microbes into the portal circulation . The gut - derived microbial products, including lipopolysaccharide and bacterial dna, can enter the liver and induce inflammation by activating toll - like receptors in kupffer cells and hepatocytes . In addition, the innate immune system is activated in the adipocytes, and the release of various adipokines (interleukin-6 and tumor necrosis factor - alpha) can contribute to hepatic inflammation . Combined with the host genetic background, the complex interplay of these mechanisms can produce inflammation, hepatocellular injury, and cell death and activate fibrogenesis . The genetic susceptibility was first demonstrated by the first genome - wide association study (gwas) on nafld in a population of hispanic, african american, and european american individuals . Variations in palatin - like phospholipase domain - containing 3 (pnpla3) were shown to influence ancestry - related and inter - individual difference in hepatic fat content and susceptibility to nafld . Specifically, the allele rs738409 encoding for the isoleucine - to - methionine variant at protein position 148 (i148 m) was strongly linked to hepatic fat accumulation and inflammation . This finding was replicated in several studies across different populations and found to be associated with nafld disease severity [1824]. Through a similar gwas strategy, other single - nucleotide polymorphisms (snps) variants near pnpla3, neurocan (ncan), and protein phosphatase 1, regulatory (inhibitor) subunit 3b (ppp1r3b) were associated with computed tomography - measured hepatic steatosis from several large - population studies . These snps were subsequently genotyped in biopsy - proven nafld from the nash clinical research network and showed that variants in or near ncan, glucokinase regulator (gckr), lysophospholipase - like 1 (lyplal1), and pnpla3, but not ppp1r3b, were associated with histologic lobular inflammation or fibrosis or both . These variants have distinct effects on various metabolic pathways indicative of genetic heterogeneity in nafld pathogenesis . More recently, kozlitina and colleagues reported the association of a transmembrane 6 superfamily member 2 (tm6sf2) variant with hepatic triglyceride content in an exome - wide association study of patients derived from the dallas heart study . The gene is more common in individuals of european ancestry (7.2%) than in african americans (3.4%) or hispanics (4.7%). The tm6sf2 variant encoding p.glu167lys correlated with elevated serum alanine aminotransferase (alt) and reductions in serum alkaline phosphatase and plasma levels of triglycerides and low - density lipoprotein cholesterol in three large cohorts . Although the exact function of the gene is unknown, it has been proposed that the tm6sf2 acts to promote very - low - density lipoprotein secretion . In another study, the tm6sf2 rs58542926 was found to be associated with nash severity and advanced liver fibrosis . Whereas nafl is characterized by the presence of hepatic steatosis with no evidence of hepatocellular injury, nash is characterized histologically by the presence of ballooned hepatocytes and lobular inflammation with or without perisinusoidal fibrosis in addition to steatosis . Diagnosing nash and although liver biopsy is considered the gold standard to diagnose and stage nash, it is invasive and has several limitations, including cost and sampling error, and is accompanied by risks (bleeding, pain, perforation, infection, and rarely death). The metabolic syndrome and diabetes are associated with increased risk for nash and can be used in selecting those patients for liver biopsy . The levels of aminotransferases are not reliable, and imaging studies have not been accurate in the diagnosis of nash and this is because the histologic features of apoptosis, ballooning, and cell injury that characterize nash are not detectable by currently available modalities . The poor performance of liver biopsy has led to significant interest in developing non - invasive biomarkers for identifying steatohepatitis or fibrosis (or both) in patients with nafld . Multiple biomarkers and predictive models have been suggested to predict nash but with varying degrees of success, and the majority require external validation . Circulating keratin 18 (ck18), a marker for hepatocyte apoptosis, is the most validated biomarker that is reproduced in several studies for differentiating nash from simple steatosis [3437]. In a recent meta - analysis, plasma ck18 levels had a sensitivity of 78%, specificity of 87%, and an area under the receiver operating curve (auroc) of 0.82 for identifying steatohepatitis . Though fairly specific, this biomarker lacks the sensitivity needed for it to be an adequate screening test to stage nash . The nafld fibrosis score and enhanced liver fibrosis (elf) panel are non - invasive tools that facilitate the identification of the majority of advanced fibrosis patients but without the need of a liver biopsy . The nafld fibrosis score is a scoring tool calculated by incorporating clinical parameters that include age, glycemia, body mass index, platelet count, albumin, and aspartate transaminase - to - alt ratio . This has been validated in many studies and confirmed by a recent meta - analysis to predict advanced fibrosis with an auroc of 0.85 . The elf combines three serum markers of matrix turnover (timp 1, hyaluronic acid, and p3np) and can identify advanced fibrosis in nash patients with an auroc of 0.87 . However, both nafld fibrosis score and elf perform poorly in early and intermediate stages . Modalities that measure liver stiffness were developed to assess liver fibrosis . Transient elastography (fibroscan) was first evaluated in a japanese study that reported a stepwise increase in liver stiffness with increased severity of liver fibrosis . A meta - analysis showed a pooled auroc of 0.94 with 94% sensitivity and 95% specificity for advanced fibrosis . A major drawback of this modality is its inconsistency in obtaining measurements in obese patients . Using a new xl probe has enabled the examination in obese patients with diagnostic accuracy comparable to that of the standard probe . More recently, studies have shown promise of acoustic radiation force impulse (arfi) sonoelastography for the assessment of liver stiffness [4446]. Aside from having the capability to be integrated in a standard liver ultrasound, this modality can evaluate large parts of the liver in one examination and is more reliable than transient elastography in obese patients . In a study of 57 patients with nafld, arfi showed a diagnostic accuracy similar to that of transient elastography for advanced fibrosis when compared with liver histology . A recent meta - analysis of various chronic liver diseases confirmed this finding, although only a small proportion of patients were in the nafld group . Another imaging modality is magnetic resonance elastography, which allows evaluation of the whole liver and shows good accuracy for detecting severity of liver fibrosis . More studies are needed to assess the role of these imaging tools in assessing changes in fibrosis in response to treatment . Meaningful therapy for nash should target reduction of mortality, development of metabolic comorbidities, liver - related and cardiovascular outcomes, and improvement of symptoms and quality of life . Despite an increasing understanding of the mechanisms of nafld pathogenesis, approved therapy is lacking . Lifestyle modifications (diet and regular exercise) remain the initial therapeutic strategy for nash . This simple yet challenging approach has been shown to correlate with nash improvement histologically [4951]. The majority of the patients with nash have concurrent metabolic comorbidities, and not surprisingly the leading cause of their mortality is cardiovascular disease . The presence of multiple features of the metabolic syndrome is associated with progressive liver disease . Therefore, regardless of nash diagnosis by liver biopsy, the treatment of obesity, diabetes, dyslipidemia, and hypertension should be optimized in nafld to reduce the risks for cardiovascular disease and liver disease progression . Pharmacologic therapy can be considered only in patients with biopsy - proven nash until reliable biomarkers are available . Insulin sensitizers, angiotensin - converting enzyme inhibitors and receptor blockers, anti - oxidants, bile acids, cytokine - mediated therapy, lipid - lowering agents, and endocannabinoid antagonists have been proposed, but the majority of these agents either lack efficacy or require further studies for validation before definitive recommendations can be made . This article will focus on pharmacologic options for which sufficient evidence is available to make a recommendation . Vitamin e is an antioxidant that suppresses lipid peroxidation, helps replete glutathione stores, decreases cytokine production, and inhibits profibrotic activity [5558]. To date, the pioglitazone versus vitamin e versus placebo for the treatment of nondiabetic patients with nonalcoholic steatohepatitis (pivens) trial is the largest randomized controlled trial comparing vitamin e (800 iu / day) and pioglitazone (30 mg / day) with placebo for 96 weeks in non - diabetic and non - cirrhotic adult patients with nash . Vitamin e was superior to placebo in improving nash histology, but no significant improvement in fibrosis was observed . There are insufficient data to recommend vitamin e for nash patients with concomitant diabetes or cirrhosis, and long - term efficacy and safety of vitamin e remain uncertain . It is important to note that vitamin e at high doses has been implicated in increased all - cause mortality . Other safety concerns of vitamin e include increased risk for development of hemorrhagic stroke and prostate cancer . Therefore, competing risks should be discussed with non - diabetic patients with nash before starting on therapy . Thiazolidinediones (tzds) are peroxisome proliferator - activated receptor - gamma agonists that ameliorate insulin resistance in adipose tissues, liver, and muscles . Tzd increases adiponectin, reduces fatty acid synthesis, and increases fatty acid oxidation by activating 5 adenosine monophosphate - activated protein kinase (ampk) and counteracts pro - inflammatory cytokines . Belfort and colleagues showed that pioglitazone improved liver histology by improving steatosis, inflammation, and ballooning in patients with nash who have impaired glucose tolerance or diabetes . In the largest clinical trial (pivens), although pioglitazone did not achieve the primary endpoint as assessed by the nafld activity score (nas), resolution of nash was achieved in a higher percentage of non - diabetic patients receiving pioglitazone versus placebo (47% versus 21%, p = 0.001). A recent meta - analysis suggested that pioglitazone may improve liver histology and fibrosis in patients with nash . In terms of safety, treatment with pioglitazone may not be without adverse effects, and its long - term use has not been studied . Potential adverse effects include weight gain, higher rate of heart failure, bone loss, and rarely bladder cancer [6770]. Overall, pioglitazone is an option in patients with biopsy - proven nash, particularly for those with impaired glucose tolerance or diabetes, but this medication must be used with caution . Obeticholic acid is a synthetic fxr agonist that has been demonstrated to decrease hepatic fat and fibrosis in animal models of nafld [7173] more recently, neuschwander - tetri and colleagues performed a placebo - controlled, randomized clinical trial (flint) that assessed the efficacy of obeticholic acid (25 mg / day) versus placebo for 72 weeks in patients with non - cirrhotic nash . The primary outcome was improvement in liver histology, defined as a decrease in nas by at least 2 points without worsening of fibrosis from baseline to the end of treatment . Treatment with obeticholic acid improved all features of the nas (steatosis, hepatocellular ballooning, and lobular inflammation) and fibrosis . However, complete resolution of nash was not achieved in a substantial proportion of patients . Although obeticholic acid was generally well tolerated, it was associated with frequent pruritus, increased serum cholesterol, and changes in the lipid profile . Therefore, long - term studies are needed to assess the potential risk of obeticholic acid for the development of atherosclerosis and cardiovascular disease in these patients . Apo c2, apolipoprotein c2; apo c3, apolipoprotein c3; fxr, farnesoid x receptor; ldl - cholesterol, low - density lipoprotein cholesterol; hdl - cholesterol, high - density lipoprotein; lxr, liver x receptor . A better understanding of nash pathogenesis may help clarify the best approach in managing patients with nash . More studies are warranted in understanding the influence of genetics associated with disease progression and patient response to treatment . Although liver biopsy is still required, biomarkers to diagnose nash and tools to predict advanced fibrosis may be helpful, but more studies are needed to validate their use . While obeticholic acid has shown promise in nash by improving not only liver histology but also fibrosis, not achieved by either vitamin e or pioglitazone, long - term studies are needed to assess the safety of obeticholic acid and its effects on liver- and cardiovascular - related outcomes . The multiplicity of pathways contributing to nash and fibrosis progression provides a rationale for individualized treatment strategies . Arun j. sanyal is a consulting advisor for abbot, genetech, gilead sciences, ikaria, merck, norgine, roche, salix, takeda pharmaceutical company, nimbus, nitto denko and bristol - myers squibb, receives research grants from ikaria, takeda pharmaceutical company, astellas, novartis and galectin therapeutics and receives royalties from uptodate inc.
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Relationships between urban air pollution and hospitalizations for cardiorespiratory causes are well established in many studies around the world [13] and in france . By comparison, published studies about the health effects of industrial air pollution on population living near industries are sparse, and few studies investigate the impact of industrial air pollution on cardiovascular or respiratory hospitalizations [57]. This paper presents the first study on the impacts of industrial air pollution on cardiorespiratory hospitalizations, in one of the largest industrial areas in france . The etang - de - berre area is a large pond (0.15 km) surrounded by three major industrial complexes gathering several oil refineries, chemical plants, ironworks, metal plants, a waste incineration plant, an airport, and the largest french seaport [8, 9]. This industrial area located in the provence - alpes - cte d'azur region has experienced a strong economic growth since the 70s . The population has doubled between 1970 and 2000, and, today, about 300,000 inhabitants are more or less exposed to the plumes of industries . The contribution of the etang - de - berre area to the regional emissions is estimated at 58% for sulfur dioxide (so2), 13% for particulate matter under 10 m (pm10), 23% for nitrogen oxide (nox), and 10% for volatile organic compounds the main sources are the industries and and the production of energy for so2 and voc emissions, industries and road traffic for pm10 emissions, and industries, production of energy, and road traffic for nox emissions . So2 concentrations measured by the air quality network in this area are still the highest observed at the regional level, even if they had decreased regularly during the last 20 years . In 2008, all monitoring stations in the area exceeded the 2005 world health organization (who) air quality guidelines for maximum daily mean concentrations (20 gm). None exceeded the european council directive 2008/50/ec of 21 may 2008 hourly limit values (hourly mean> 350 gm / more than one day). Pm10 concentrations are relatively stable 10 years ago, but some peaks are still measured . In 2008, all the monitoring stations exceeded the who air quality guidelines (annual mean of 20 gm). Nitrogen oxides (nox), heavy metals, and polycyclic aromatic hydrocarbons (pah) concentrations were under the 2008/50/ec limit value, whereas benzene concentrations were slightly higher near the industrial sites . Ozone concentrations were high in summer because of the emissions of ozone precursors and the high degree of sunshine but this affects all the regional area . Since the 1990s, environmental protection associations created by the population request an assessment of the health of population living near these polluting and potentially dangerous industries the administrative authorities decided to carry out quantitative health risk assessments (hra), based on the comparison of exposure to pollutants with toxicological reference values (trv), for the three main industrial complexes between 2006 and 2011 . The first hra, on the oil refining area of berre - l'etang, began in 2006 and revealed high benzene and 1.3 butadiene fugitive emissions at the refinery . Carcinogenic risks by inhalation exposure were found above the reference threshold of 10 for the population living in the city of berre - l'etang and in a large northern part of the study area . Corrective measures to reduce emissions of these two compounds an updated hra carried out in 2008 showed a decrease of the area exposed to benzene, from 30 km to 10 km around the industrial site . Yet, carcinogenic risks by inhalation exposure were still above the reference threshold of 10 for the population living in the north part of the study area . An hra on the industrial - port area of fos - sur - mer found that so2 and pm10 modeled concentrations were higher than the air quality guidelines in all the study area . Carcinogenic risks by inhalation exposure were under the reference threshold of 10 for the entire population living near the industrial site . The last hra on the petrochemical area of lavra - la mde found that so2 and pm10 modeled concentrations were higher than the air quality guidelines in all the study area . Carcinogenic risks by inhalation exposure were above the limit threshold of 10 for the population living in a part of the study area representing 21,000 inhabitants . These studies have led to a complete inventory of the different pollutants emitted by the industries and have helped prioritizing actions to reduce the exposure of the population . So2 and pm10 pollutants were classified as requiring priority actions to reduce industrial emissions and population exposure, although it was not possible to assess the related health risks in the hra, as tvr are not available for these compounds . Decreasing benzene, 1,3-butadiene, chrome vi, and 1,2-dichloroethane industrial emissions was also recommended to decrease the exposure of workers and of the population neighboring the industrial sites . However, these studies cannot answer the main concern of the population: is the health of the people living in this industrial area worse than the health of people living in nonindustrial areas? Therefore, the administrative authorities asked the regional office of the french institute for public health surveillance to carry out an epidemiological study . After a review of the existing studies and of the routinely available data for this area, we decided to conduct an ecological study on hospitalizations data . The objective of this ecological study was to estimate a relationship between hospitalizations ratios and so2 exposure levels at the district of residence . Comparison was done between exposed and nonexposed district, controlling on socioeconomic status estimated through townsend's index and proportion of male workers in each district, which are factors potentially influencing people health and exposure . The study area is located in the provence - alpes - cte - d'azur region near the mediterranean sea . Its boundaries were based on modeled so2 concentrations, topographic criteria, and labour pool . It included 29 administrative districts (named districts afterwards) surrounding the etang - de - berre pond and represented 399,962 inhabitants living on a 975 km area (figure 1). Almost 50 of them have dangerous activities related to a high risk of industrial accident and are classified as high threshold according to the european council directive 96/82/ec of 9 december 1996 on the control of major - accident hazards involving dangerous substances . These industries are grouped in 3 main complexes (figure 2):the lavera - la mde area located in the district of martigues, operating oil refining, petrochemical and organic chemical activities, and chlorine chemistry since the 1950s;the berre area located in the district of berre - l'etang operating oil storage and petrochemical industry . The first refinery was settled in 1933; the industrial port area of fos - sur - mer including steel and metal working, chemicals plants, waste incineration plant, and the port for ore and oil tankers settled since the 1970s.the etang - de - berre area is also crossed by a dense road network which supports a high traffic of heavy trucks related to the industrial and harbor facilities and of passenger cars commuting from home to work . The lavera - la mde area located in the district of martigues, operating oil refining, petrochemical and organic chemical activities, and chlorine chemistry since the 1950s; the berre area located in the district of berre - l'etang operating oil storage and petrochemical industry . The first refinery was settled in 1933; the industrial port area of fos - sur - mer including steel and metal working, chemicals plants, waste incineration plant, and the port for ore and oil tankers settled since the 1970s . The local air quality network (air paca) measures air pollution levels since 1972 . In 2008, 27 monitoring stations settled on the study area (figure 1) measured continuously the following pollutants: sulfur dioxide (so2), ozone (o3), nitrogen dioxide (no2), particulate matter (pm10), carbon monoxide (co), and benzene . For so2 concentrations in the study area, annual mean levels of the different monitoring stations varied between 2 and 18 g m and maximum hourly mean levels between 83 and 831 gm (table 1). The highest values are measured by the industrial monitoring stations . In comparison, for the 6 stations located on the rest of the region, annual mean levels varied between 1 and 4 g m and maximum hourly mean levels between 20 and 132 gm . Exposure to air pollution was assessed at the district level, using so2 concentrations as a proxy for industrial emissions . Air paca provided the mean concentrations of so2 for 2008 on a 200 m200 m grid using a dispersion model (adms4), a meteorological model and kriging . We used a geographic information system (gis) to assign a concentration level to each district . To aggregate concentrations data, urban areas of each district were identified based on the 2006 land cover classification system . Urban areas included urbanized areas, major roads and railways, commercial, industrial, and working areas, leisure activities areas, and public gardens . For each district, the concentrations were averaged weighted on the cells proportion included in urban areas as illustrated in figure 3 . The annual mean levels of so2 varied between 2.1 and 12.4 gm depending on the district (figure 4) and were grouped in three classes of exposure based on quartiles: reference (<4.2 gm); medium (between 4.2 and 6.4 gm), and high (> 6.4 gm) values (table 2). Reference levels were similar to the so2 annual mean levels measured in nonindustrial districts in the regional area, varying from 1 to 4 gm . We also investigated whether pm10 concentrations could be an industrial pollution indicator for this ecological study . Annual mean levels of the different monitoring stations varied between 27 and 33 gm in the study area and were similar to those measured in the rest of the region (table 3). With the same method used for so2, the estimated annual mean levels of pm10 varied between 27.8 and 33.6 gm depending on the district . The spatial distribution of concentrations was relatively homogenous and the highest concentrations were not observed at industrial districts . The french programme for hospital information system (pmsi) is implemented since 1994 in public hospitals and since 1997 in private hospitals . It is a medical economic database based on the diagnosis - related group (drg) method . Each hospitalization is registered in a local database grouped in a national database . Since 2004, a patient identification number is included to identify patients and hospital stays related to each patient . The national hospitalization database held by the pmsi provided hospitalization data for the whole region . Hospital stays included in the analysis were selected over the study period 20042007 based on several selection criteria . On the first step, we excluded stays without patient identification number and stays for patient that moved outside or inside the study area between 2004 and 2007 . On the second step, stays for the studied diseases were selected from the main diagnosis at the discharge, coded with the 10th revision of the international classification of diseases (icd-10), and sometimes from secondary diagnosis . The first hospitalization of each resident over the study period was retained in order to approximate a hospitalization incidence for each health indicator . Respiratory and cardiovascular hospitalization indicators have been selected from the papers on links between air pollution and health [1527]. The selection of cancer hospitalization indicators was based on knowledge about frequencies of different type of cancer at the regional level and on the results of two french reports on environmental cancers [28, 29]. The following hospitalization indicators were defined: all cardiovascular diseases (icd-10: i00i99), heart diseases (icd-10: i00i52), and coronary heart diseases (icd-10: i20i24), myocardial infarction (icd-10: i21-i22), stroke (icd-10: i60i64 or g45-g46), heart rate disorders (icd-10: i44i49), coronary heart diseases with heart rate disorders (icd-10: i20i24 as main diagnosis and i44i49 as secondary diagnosis);all respiratory diseases (icd-10: j00j99), respiratory infections (icd-10: j04j06 or j10j18 or j20j22), pneumonia (icd-10: j10j18), asthma (icd10: j45-j46), and exacerbations of chronic obstructive pulmonary diseases (principal indicator algorithm described in); all cancers (icd10: c00c97), lung cancer (icd10: c33-c34), bladder cancer (icd10: c97), breast cancer (icd10: c50), multiple myeloma (icd10: c90), malignant non - hodgkin's lymphoma (icd10: c82c85), and acute leukemia (icd10: c910, c920, c924, c925, c930, c942, c943, c950). All cardiovascular diseases (icd-10: i00i99), heart diseases (icd-10: i00i52), and coronary heart diseases (icd-10: i20i24), myocardial infarction (icd-10: i21-i22), stroke (icd-10: i60i64 or g45-g46), heart rate disorders (icd-10: i44i49), coronary heart diseases with heart rate disorders (icd-10: i20i24 as main diagnosis and i44i49 as secondary diagnosis); all respiratory diseases (icd-10: j00j99), respiratory infections (icd-10: j04j06 or j10j18 or j20j22), pneumonia (icd-10: j10j18), asthma (icd10: j45-j46), and exacerbations of chronic obstructive pulmonary diseases (principal indicator algorithm described in); all cancers (icd10: c00c97), lung cancer (icd10: c33-c34), bladder cancer (icd10: c97), breast cancer (icd10: c50), multiple myeloma (icd10: c90), malignant non - hodgkin's lymphoma (icd10: c82c85), and acute leukemia (icd10: c910, c920, c924, c925, c930, c942, c943, c950). The number of hospitalizations selected for the study area represented 9% of the cardiovascular and respiratory diseases hospitalizations and 7% for cancers hospitalizations registered at the regional level . The 2006 national census held by the french national institute for statistics and economic studies (insee) provided data on socio - occupational groups of the working population in the study area and for the socioeconomic items included in townsend's index . This index was built using the following socioeconomic items: proportion of unemployed person among working population, proportion of main homes with more than one person per room, proportion of main homes occupied by not owner household, and proportion of household without a car . Standardized socioeconomic variables using regional values as reference were used to build an additive scale for each district . The proportion of male workers was retained as a confounding factor, making the hypothesis that it would be a good predictor of the industrialization of each district . We calculated the expected number of cases at the district level for each health indicator by standardization method using the regional population as reference . Then standardized hospitalization ratios (shr) were calculated as the rate of observed to expected cases . Relative risks of hospitalization for people living in medium or high exposed districts were calculated compared to those living in the reference districts . Overdispersed poisson regression models were fitted to assess the association between hospitalization ratios and classes of exposure to industrial pollution, taking into account potential confounding factors . The second level splits the residual risk into a linear combination of covariate effects xti and into random effects ui and vi measuring excess heterogeneity and spatial similarity, respectively: (1)log(i)=+xti+ui+vi, where the term exp() is the overall relative risks of disease in the study area compared to the reference rate . The vectors u and v are supposed independent, and u, that models the excess heterogeneity of the relative risks, is assumed to follow a normal distribution ui ~ n (0, u). To model spatial similarity in residuals the gaussian conditional autoregressive model (car) is used as the prior for the spatial component v: (2)(vivj= i, ji)~n(jiwijvjjiwij,v2jiwij), where the wijs denote weights defining which districts j are neighbors to district i (by convention wii = 0 for all i). We used the adjacency - based weights where wij = 1 if district j is adjacent to district i, wij = 0 otherwise are used . We have taken gamma prior distributions for the precision parameters (reciprocal of the variance) of the heterogeneity and spatial terms . For both the (a, b) denotes the gamma distribution with expectation equal to a / b . Non - informative priors were taken for the other parameters, that is, the intercept and the regression coefficients . In a bayesian context, we defined the credible interval at the 5%, that is, the probability that the parameter belongs to is 95% . Analysis was done by age (children 014 years, adults over 15 years) and by sex for the adults with the software r and winbugs . The highest so2 levels (> 6.4 gm) were observed in the highly industrialized districts in the south of the etang - de - berre area . Districts in the northeast of the study area had the lowest levels of air pollution (figure 3). High values are related to a low socioeconomic status (ses) and negative values to a rather high ses . Districts in the north of the study area were rather favored and industrial districts rather deprived . This index was significantly correlated with the socio - occupational group but moderately with so2 exposure levels (coefficient of correlation = 0.4). For all indicators, the number of cases varied between districts depending on the population size . The sex ratio male / female varied from 1.2 for all cardiovascular diseases to 2.4 for myocardial infarction (mi) hospitalizations . Hospitalizations for exacerbations of copd occurred rather in males (sex ratio = 2.5) while hospitalizations for respiratory infections, pneumonia, or asthma occurred in both sex (sex ratio from 1 to 1.2). Men were more hospitalized for acute leukemia, lung, and bladder cancer (sex ratio at 1.5, 3.3, and 5.0, resp . ). Children accounted for half of the patients hospitalized for asthma, one third for respiratory infections and 15% for pneumonia . On the other hand, children accounted for less than 1% of the patients hospitalized for cardiovascular diseases or cancer . For children, the risk to be hospitalized for respiratory conditions was the same in the high or medium exposed districts and in the reference districts . The risk was slightly increased in districts with low socioeconomic status (table 5). For adults, and for most of the studied indicators, the risk to be hospitalized was the same in areas with medium or high exposure to industrial air pollution and in areas exposed to reference levels . However, the relative risk (rr) to be hospitalized for an acute leukaemia increased significantly to 2.6 for men living in districts with high so2 levels . We found a significant increase of the risk to be hospitalized for myocardial infarction in the districts exposed to industrial air pollution, especially in women (table 6). Excess risk to be hospitalized for mi in women living in districts with medium or high so2 exposure was, respectively, 38% [ci 95% 4%: 83%] and 54% [14%: a 26% [2%: 57%] excess risk to be hospitalized for mi was observed in men living in districts with high so2 levels only compared to those living in districts at reference levels . This is the first ecological study on hospitalizations related to industrial air pollution near a large industrial estate in france . An excess risk of hospitalizations for myocardial infarction was found for women living in the districts exposed to industrial air pollution and for men living in the highly exposed districts . These results are similar to those reported by fung et al . In a canadian study, where shr for cardiovascular and respiratory diseases increased in industrial cities compared to a reference city, with higher ratios in women . On the other hand, a study set in england and wales did not show any excess risk of hospitalization for cardiovascular, cerebrovascular, and respiratory diseases among the population living near coke works . The estimated excess risk of hospitalizations for acute mi was greater in women while men were mostly hospitalized for cardiovascular causes . This could be related to a higher sensitivity of women to the effects of air pollution or to a better control of confounding factors in men than in women . A local study showed a correlation between the socio - occupational group and smoking in men . Daily smoking is twice as much common for workers and unemployed persons than for managers . So, the adjustment of the analysis on the proportion of male workers allowed us to control partially smoking in men but not in women . We did not find excess risk for asthma hospitalizations in children while a case crossover study found a relationship between hospitalizations or emergency visits for asthma attack and so2 peaks in children living near refineries (no association was found when using so2 daily means). The lack of significant results for respiratory diseases most probably shows that hospitalization indicators are not the best indicators to evaluate the respiratory health effects of air pollution in adults in france . Asthma hospitalization rate in adults decreased slightly since 2000, and asthma disease is mostly taken care of by ambulatory management . Studies using emergency or general practitioner (gp) visits for asthma attacks could be more relevant . Moreover most of the published studies concern the analysis of asthma or respiratory symptoms prevalence in children living near industrial sites by comparison to those living in a nonexposed area [3638]. Pulmonary function tests found a decrease in lung function and an increase of airway inflammation . Regarding cancer, results reflect past exposure because of the long latency period between exposure and onset of cancer . It would have been much better to estimate patient's exposure 1015 years ago but we had no information on their place of residence before the hospitalization . Only one significant association was found between the exposure to industrial air pollution and acute leukemia in men . This result must be considered with caution because of the small number of observed cases . However, this association observed in men may suggest a potential occupational exposure due to compounds processed or emitted by petrochemical industries . Some of them are classified as carcinogenic for human (benzene, 1.3-butadiene) or likely carcinogenic for human (1.2-dichloroethane), and benzene is commonly considered as a risk factor for acute myeloid leukemia [39, 40]. This hypothesis needs to be evaluated by local studies on the occupational exposure to these carcinogenic compounds . The strength of this study was the estimation of the exposure to industrial air pollution using modeled so2 concentrations rather than a distance to the industrial source . This pollutant was the best proxy of industrial air pollution as industrial sources provided 85% of the total so2 emissions in the study area . Annual mean concentrations of so2 were used in this study rather than hourly values for practical reasons and time consuming . Anyway, monitoring stations with the highest annual means were those with the hourly values too . Using so2 annual mean to model industrial air pollution rather than hourly values should not change the class of exposure of each district . Particulate matter (pm10) concentrations were emitted by many other sources, than industrial sources and could not identify correctly industrial districts . In fact, as shown by the three hra studies, many pollutants other than so2 are emitted by industries in particular particles . Several studies have shown short - term effects of particulate matter (pm) on hospital admissions from cardiovascular causes [1522], and myocardial infarctions have been shown to be susceptible to being triggered by pm [1619]. Population living near industries is exposed to a mixture of pollutants, and particles could play a role in the observed excess of myocardial infarction hospitalizations . In our study, exposure to air pollution, assessed as the annual average levels of modeled concentrations, depends on the parameters of dispersion and meteorological models . Using average values for each geographical unit may have resulted in a dilution effect of exposure when modeled concentrations were heterogeneous within districts . We limited this dilution effect by computing the average concentrations only in the urban area, making the hypothesis that people spent most of the time in this area during the day . In ecological studies, the choice of exposed and non - exposed areas is usually based on the distance to the industrial site, making the hypothesis that exposure decreases as the distance increases [4144] whereas a set estimation of exposure would be more relevant . One study used an approach based on so2 and nitrogen dioxides (nox) levels, taking only into account levels above limit values . Pollutant levels were interpolated by kriging, and a gis was used to assign a mean concentration at residential address to each case . Another study used gis tools to assign an individual integrated score of exposure that accounts for subject's mobility, length of residential stay, distance to petrochemical plants, wind direction, and industrial pollution sources . However, these studies were cross - sectional studies based on individual data and none of them used aggregated health data . Regarding the design of our study, the main advantage of ecological studies is the use of aggregated data which are often routinely produced, such as hospitalization data . These data are potentially biased by coding or ranking errors that are not differential and lead rather to an underestimation of the relationship with air pollution exposure . The main error of ecological studies is the ecological bias related to heterogeneity in the geographical units due to one or more uncontrolled confounding factors that could be related to exposure and/or to health indicators . Our models are adjusted on the socioeconomic status estimated by the index of townsend and the proportion of male workers in the working population . For this local study, the index of townsend distinguishes relatively well between the industrialized districts and the favored residential municipalities but is more variable in districts under plumes of industries ., fos - sur - mer is an industrial highly polluted district but is situated in the middle class for ses . In the literature, studies carried out on links between social deprivation, health, and air pollution use either several socioeconomic items (average annual income, proportion of people below the poverty threshold, educational level, proportion of unemployed person, proportion of workers, and marital status) or synthetic index of deprivation as those of townsend, carstairs and morris, or jarman . Sometimes, synthetic indexes are specifically built from several socioeconomic variables either by a factorial [49, 50] or by an additive approach [51, 52]. These specific indexes, more representative of the local deprivation, are often used to analyze the ses modifying effect on pollution exposure . In our study we used the deprivation index as confounding factor, and townsend's index seemed to estimate correctly deprivation at district level as reported by declercq and prouvost . Determinants of the healthcare system can also potentially modify the relationship between exposure and hospitalizations . In france, access to healthcare is available for the quasi - totality of the population, and the very few access restrictions do not constitute a real limit in our study . On the other hand, the use of health care is linked to the socioeconomic status of the patients and to the socioeconomic context of residence . We did not control directly the possible heterogeneity in the use of health care because of the lack of available data at district level . However, it was indirectly taken into account through the index of townsend and by the bayesian hierarchical model controlling the spatial autocorrelation . This modeling allowed us to limit the bias due to variability in use of health care between districts . Finally, in the ecological studies, the individual confounding factors such as obesity, cholesterol level, lifestyle, smoking, and alcoholism cannot be taken into account because of using aggregated data at district level . This study underlines that, in terms of hospitalizations for respiratory diseases and cancers, the health condition of the population exposed to the industrial air pollution was similar to those of nonexposed people . However, the results illustrate the impact of industrial air pollution on the cardiovascular system . Efforts should be done to decrease the levels of so2, particles, and some carcinogenic compounds emitted by the industries, by improving industrial processes and using less polluted fuels . For instance, decreasing the level of road traffic particles would require the implementation of an interurban public transport network, as well as the development of rail transport for raw materials and goods . Prevention of the cardiovascular diseases should be a public health priority in the study area, particularly in women . General practitioners, key players in the health prevention, would have clear and useful information on harmful cardiovascular effects of air pollution . Finally, occupational medicine should reinforce the screening of hematopoietic disorders, myelodysplasia, and acute leukaemia in workers as well as in pensioners of refineries and petrochemical plants.
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Red blood cell (rbc) units are typically available for transfusion as early as 3 - 4 days after collection and with modern preservation techniques solutions can be administered up to 42 days after collection . Despite these milestones of transfusion medicine, these reversible and irreversible changes, known as storage lesion, start in about two to three weeks of storage - time . During storage, erythrocytes undergo corpuscular changes, including depletion of 2,3-diphosphoglycerate and adenosine triphosphate, increased rigidity, a progressive depletion of nitric oxide, and a significant increase in abnormally shaped rbcs [1, 3,4,5,6,7]. Furthermore, cytokines that accumulate with storage of rbcs are associated with transfusion - mediated systemic inflammatory reactions and higher risk of bacterial infections . Many studies propose that the amount and the storage time of transfused rbc units are associated with reduced survival and significantly increased risk of postoperative complications like prolonged intubation, renal failure, septicaemia or sepsis, multi - organ failure, and other serious complications [1, 4, 9,10,11,12,13,14,15]. In postoperative patients (above all in cardiac surgery patients) there are many particular situations (like low cardiac index, hypovolemia, long cardiopulmonary circulation and atrial fibrillation) which cause compromised renal function . In that moment the concomitant rbc transfusion further worsen renal function . Therefore, it is mandatory to analyse specific organ function . The extent of perioperative renal impairment ranges from subclinical injury to established renal failure requiring dialysis . Acute renal failure affects 1 - 5% of cardiac surgery patients and remains a major cause of morbidity and mortality [3, 8, 16,17,18,19,20,21,22,23,24,25]. Therefore, the purpose of this retrospective observational cohort study was to focus on the impact of storage time and amount of transfused red blood cells on renal function . In the period between january 2009 and january 2010, 961 patients underwent cardiac surgery at the clinic for thoracic, cardiac and vascular surgery of wrzburg university hospital and 492 (51.2%) were transfused . The following perioperative data were collected: sex, age, body mass index, emergency operation, diabetes mellitus, chronic obstructive pulmonary disease, operation type, cardio - pulmonary bypass time, aortic - cross - clamp time, storage time of rbcs in days, amount of transfused rbcs, serum creatinine, glomerular filtration rate (gfr) and urea starting preoperatively until postoperative day two . Patients were pooled in different groupsdepending on storage time (group 1 - group 6) and amount of rbc transfusions (group 7 and 8). Group 1 (mean age of all transfused rbcs 14 days); group 2 (mean age of all transfused rbcs> than 14 days); group 3 (only one rbc unit was transfused, age 14 days); group 4 (only one rbc unit was transfused, age> 14 days); group 5 (only rbcs older than14 days); group 6 (only rbcs younger than 14 days); group 7 (five or less rbc units, regardless of storage time); group 8 (more than 5 rbc units, regardless of storage time). All data were taken retrospectively from the central hospital computer and the blood - bank database . The following inclusion criteria were defined: male or female patients aged 18 years or older, transfusion of at least one (or more) buffy - coat depleted red blood cell unit, and cardiac surgery (coronary - artery bypass grafting (cabg), valve surgery, or a combination of cabg and valve surgery, or surgery on the thoracic aorta). Exclusion criteria were defined as follows: immunosuppressive therapy, concurrent immunological disease, and preoperative signs of inflammation . The management of transfusion (transfusion - trigger) was defined as a haemoglobin value <8.0 g / dl in combination with at least one of the following: urine output <100 ml / hour, and/or svo2 <65%, and/or lactate> 3.0 mmol / l, and/or pao2 <60 mmhg or sao2 <90% . Obtaining approval of the local ethics committee was not applicable due to the retrospective nature of the study . Definitions . Kidney insufficiency and kidney failure were defined according to society of thoracic surgery (www.sts.org) itemized on the national database definition clarification of the sts . Acute kidney insufficiency was defined as serum creatinine values greater than 2.0 mg / dl or a duplication of the preoperative value (baseline). Pathological glomerular filtration rate (gfr) was defined as less than 60 ml / min . Pathological urea value was defined as greater than 50 mg / dl . Statistical analysis . The results were summarized in a database and evaluated with the statistic program spss (18.0). A p - value <0.05 was deemed to be statistically significant . Descriptive statistics with continuous variables are reported as numbers and percentages of answered questions for categorical variables . A group overview was also prepared for nominal - scaled variables . To determine differences in these values, the chi - square test of independence was performed . For ratio - scaled variables a descriptive overview of the eight groups the eight groups were then analysed with the mann - whitney u test for significant differences . If the variable was binomial (e.g. Gender) fisher s exact test was calculated . Data were processed and analysed while preserving anonymity . For each combination of groups and the examined parameter we performed a chi square test according to pearson . During the study period 492 consecutive transfused patients, aged of 6812.8 years, 169/492 (34%) females, fulfilled the inclusion criteria and were involved in the analysis . Altogether, 2,133 rbc units were transfused with a mean storage time of 21.87 days . The mean age of rbcs according to the different groups is shown in table 1 . The percentage of patients receiving 1, 2, 3, 4, 5 rbc units or more than 5 rbc units is shown in figure 1 . Percentage of patients receiving 1, 2, 3, 4, 5 red blood cell units or more than 5 . Two rbc units were transfused in 128/492 (26%) patients while 114/492 (23.2%) patients received more than 5 rbc units and 29/492 patients (5.9%) received more than 10 rbc units . Groups were well matched with regard to pre and intraoperative data (table 2). Preoperative serum creatinine, gfr, and urea values according to the different groups are shown in table 3 . Preoperative serum creatinine, glomerular filtration rate, and urea values according to the different groups . On the second postoperative day, the mean valve of creatinine, gfr, and urea was 1.45 mg / dl, 58.96 ml / min, and 48.74 mg / dl respectively . There was a significant correlation of postoperative pathological serum creatinine (p<0.01), gfr (p<0.01), and urea (p<0.01) with the amount of transfused rbcs (group 7 vs 8) but not with the storage time (comparing groups 1 - 6) (table 4). Postoperative pathological serum creatinine, glomerular filtration rate, and urea according to the different groups . Of 492 patients, 141 (29%) developed acute kidney insufficiency and 47 (9.6%) acute kidney failure . Concerning acute kidney insufficiency, there was a significant difference between group 1 and group 2 (p = 0.042), group 5 and group 6 (p=0.028), and between group 7 and group 8 (p <0.01). Acute kidney failure (need for dialysis) was performed in 47/492 patients (9.6%). Concerning acute kidney failure, there was a significant difference only between group 7 and 8 (p <0.01) (table 5). The most important finding of this study is to confirm that worsening of renal function after cardiac surgery is associated with storage time and amount of transfused red blood cell units . Complications like increased hospital stay, prolonged intubation (period) or an increase in mortality and morbidity rates are the dominating end - points in these trials . In this retrospective observational cohort study, 492 consecutive transfused cardiac surgery patients were analysed with regard to postoperative renal function . The pathophysiology of renal injury is multifactorial and is related to perioperative renal hypoperfusion and the presence of endogenous and exogenous nephrotoxins (e.g. Rbcs) and microembolism . Some previous studies support the hypothesis that the duration of storage and the amount of transfused rbc units is associated with morbidity and mortality [1, 4, 9,10,11,12,13,14,15]. Koch et al . Examined 6,002 standard cardiac surgical patients (2,872 patients received 8,802 rbcs that had been stored for 14 days or less, and 3,130 patients received 10,782 rbc units that had been stored for more than 14 days). Patients who were given older rbcs had higher rates of in - hospital mortality (2.8% vs 1.7%, p=0.004), intubation beyond 72 hours (9.7% vs 5.6%, p<0.001), renal failure (2.7% vs 1.6%, p = 0.003), and sepsis or septicemia (4.0% vs 2.8%, p=0.01). Even 1 year mortality was significantly lower in patients given newer blood (7.4% vs 11.0%, p<0.001). Furthermore, koch et al . Published three other studies showing the negative effect of rbc transfusion [4, 9, 11]. Summarizing these studies, data showed that transfusion of rbcs was associated with a risk - adjusted reduction in survival for early (defined as until 6 months postoperatively, p<0.0001) and late clinical parameters, like renal failure (p<0.001), prolonged ventilatory support (p<0.0001), serious infection (p<0.0001), cardiac complications (p<0.0001), and neurologic events (p<0.0001) were increased if rbcs were transfused . Van straten et al . Focused on the impact of the amount of rbc transfusions on mortality in a retrospective study with 10,626 cabg patients . The authors concluded that transfusion of rbc is an independent and dose - dependent risk factor for early mortality after revascularisation (p<0.001). Even secondary end - points like renal failure (p<0.001), mediastinitis (p<0.001) and perioperative myocardial infarction (p<0.001) were significant dose - dependent risk factors . In our retrospective observational study we showed that postoperative pathological laboratory findings of serum creatinine (p<0.01), glomerular filtration rate (p<0.01), and urea (p<0.01) had a significant correlation with the amount of transfused rbcs, but not with storage time . The onset of acute kidney insufficiency and acute kidney failure was correlated to storage time (p=0.042, p=0.028), and to the amount of transfused rbc units (p after adjustment for patient and disease characteristics, patients exposed to 1 or 2 rbc units had a 16% higher long - term mortality risk (adjusted hazard ratio=1.16, 95% ci: 1.01 - 1.34, p=0.035). Highlighted the association of bacterial infection with rbc transfusion in a prospective cohort study with 533 cabg patients . After adjusting for patient and disease characteristics, invasive treatments, surgical time, and the transfusion of other substances, the (adjusted) rates of bacterial infection were 4.8% with no rbc transfusion, 15.2% with one or two units, 22.1% with three to five units, and 29% with to six units (p<0.001). Even murphy et al . Showed that rbc transfusion was strongly associated with infection, and a strong dose - response relationship was present (odds ratio 3.38; 95% confidence interval, 2.60 to 4.40). Postoperative infections after allogenic blood transfusions have been attributed to transfused white blood cells in red blood cell components . In a prospective observational study with 1,553 elective and emergency cardiac surgery patients vymazal et al . Described the relationship between sternal dehiscence (following cardiac surgery) and the total number of packed red blood cells . On average, 7.6 transfusion units of allogenic blood were administered to patients with sternal dehiscence versus 1.6 (transfused units of allogenic blood) to patients without sternal dehiscence (p<0.00005). Investigated the effect of leukocyte reduction of transfused red blood cells in patients undergoing cardiac surgery (782 patients received buffy - coat - depleted rbcs before leukocyte reduction versus 632 patients with leukocyte - reduced rbcs after leukocyte reduction). The authors could not find a beneficial effect of the universal leukocyte reduction on infection (p=0.19), 90-day mortality (p=0.28), and the length of intensive care unit stay (p=0.34). These results are even demonstrated by murphy et al . And bilgin et al . Who investigated, in a double - blinded randomized controlled trial, the effect of leukocyte - depleted rbc transfusion in cardiac valve surgery . Studies denying an association between rbc transfusion and morbidity and mortality.in contrast to these data, other studies did not corroborate the previously reported association between transfusion of old rbcs and morbidity and mortality [3, 8, 17,18,19,20,21,22, 26]. In a retrospective analysis of 6,994 noncardiac surgical patients receiving transfusions during of general surgery saager et al . Reported no association between increasing median storage duration time and postoperative mortality (hazard ratio, 0.99 [0.94 - 1.04]; p=0.64). Compared 950 cardiac surgical patients who had received only rbcs older than a median storage time of 18 days (median, 24 days) with 945 patients who had received the same number of rbcs all fresher than 18 days (median, 13 days). In the multivariate analyses no significant correlation of storage time variables with 30-day survival, hospital stay, and intensive care unit (icu) stay was seen . However, the number of transfusions were independently correlated with mortality and icu stay (p<0.001). Even vamvakas et al . Has related, in 268 consecutive cabg patients, the postoperative length of hospitalization (p>0.50), the postoperative length of stay in the icu (p>0.50), and the length of endotracheal intubation (p=0.61) with the length of storage of the oldest transfused rbc units, the mean length of storage of the oldest and second oldest rbc units, and the mean length of storage of all rbc units transfused to each patient . All clinical parameters were without a significant association after adjustment for the effects of confounding factors . The authors could not find any correlation between the duration of storage of rbc and a prolonged stay in the icu, mechanical ventilation time, increased rates of perioperative infarction, mediastinitis, or sepsis . Despite missing significance, the multivariate analyses figured out that each day of storage of the oldest unit was associated with an increment of 6% of the risk of pneumonia (95% confidence interval, 1 - 11; p=0.018). The cut - off point of maximum sensitivity and specificity (54.8 and 66.9%) associated with a greater risk for pneumonia corresponded to 28 days of storage for the oldest unit (odds ratio, 2.74; 95% confidence interval, 1.18 - 6.36, p=0.019). Similar results were achieved by yap et al . Who studied 670 consecutive non - emergency cardiac surgery patients in a retrospective observational cohort study . The authors reported that the storage age of rbcs was not independently associated with postoperative early mortality (p=0.89), renal failure (p=0.16), pneumonia (p=0.47), icu stay (p=0.79), and ventilation hours (p=0.09). Van de watering performed a meta - analysis evaluating (publications of) clinical studies comparing storage time of transfused rbcs with physiological or clinical outcomes . The authors found sixteen observational studies comparing clinical outcome that yielded contradictory results regarding the effect of rbcs on mortality, length of intensive care unit and hospital stay, infections, organ failure, and composite adverse effects . Summarizing the results of the selected studies, van de watering et al . Concluded that available studies provide no evidence that longer stored rbcs are more harmful than younger rbcs . The differences between the published results may be explainable by the different study designs (retrospective vs prospective and observational vs randomized), different study populations [critically - ill patients vs less severely - ill patients, only cabg patients vs all cardiac surgery patients, patients excluded due to being expired during the first few days after surgery], different definitions of old and fresh rbcs [14 days vs 8 days], and different transfusion triggers . Hardy et al . Summarized this sophisticated issue of rbc transfusion and its clinical consequences as follows some patients are helped, for others it has no impact and for some it is harmful . The most important limitation of the present study is the retrospective observational design, which may have led to bias in confounding parameters among the different groups in contrast to a randomized design . A study using a rare resource such as blood transfusion has difficulties in being blinded and randomized . In an ongoing observational study we are analyzing patient data from 500 consecutive transfused cardiac surgery patients versus a cohort of 500 consecutive not - transfused (matched pair) patients . Transfusion of rbcs was associated with postoperative increased serum creatinine and urea, decreased glomerular filtration rate, acute kidney insufficiency, and acute kidney failure . The storage time was associated with acute kidney insufficiency, but not with acute kidney failure . Based on these findings, on recent international literature, and on the increasing problem of distributing the rare resource of human blood, it is mandatory to develop an evidence - based transfusion trigger for cardiac surgery patients.
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Bilateral carotid artery occlusion accompanied with basilar tip aneurysm is extremely rare.15) it has been suggested that increased blood flow through the vertebrobasilar system, which is the major source of collateral circulation, intensifies the hemodynamic stress on the arterial walls and this may result in the formation of saccular aneurysms.13) direct clipping for these aneurysms is difficult and the morbidity is high due to the extensive collateral networks as well as the already compromised cerebral blood flow and high internal blood pressure.2)3)7)14) thanks to the development of endovascular techniques, the aneurysms that are difficult to reach by craniotomy can now be treated with excellent results.3)4)6) to the best of our knowledge, there have been only seven cases of bilateral carotid artery occlusion with concurrent basilar tip aneurysm not being related to moyamoya disease.5)7)8)11)12)17)18) among them, only three cases were treated with coil embolization.7)12)17) we report here on a rare case of a ruptured basilar tip aneurysm that was successfully treated with coil embolization in the bilateral cervical internal carotid artery (ica) occlusions with abnormal vascular networks from the posterior circulation . A 43-year old man with a sudden onset of headache, nausea and vomiting visited the emergency room at our institution . His aunt suffered from moyamoya disease, but he had not undergone diagnostic work - up for familial cerebrovascular diseases because he had not experienced neurological deficits such as ischemic attacks, including dysarthria, dizziness and gait disturbances . Mentally alert, the patient had meningismus, experiencing neck stiffness on admission . A brain computed tomography (ct) scan demonstrated diffuse and thick subarachnoid hemorrhage (sah) in the bilateral sylvian fissures, the anterior interhemispheric fissure, the basal cistern and the posterior fossa, including the perimesencephalic region (fig . Subsequent carotid angiography showed complete occlusion of both icas, the so called' bottle neck appearance' or' beak shape,' at the level of the carotid bulb without appreciable intracranial filling and there were small collaterals from both external carotid arteries through the middle meningeal arteries to the anterior cranial fossa (fig . Vertebral artery angiography demonstrated that both hemispheres were seen filling in an antegrade fashion from the upper basilar artery and both posterior cerebral arteries through an extensive network of collateral vessels to the ica bifurcations . A 6 4 mm aneurysm was also observed at the basilar artery bifurcation (fig . Perfusion ct scans revealed no reduction of cerebral blood flow and normal cerebrovascular reactivity to acetazolamide (fig . 4). The abdominal ct aortograms did not demonstrate any other extracranial pathological vascular findings, including renal artery stenosis and fibromuscular dysplasia (figures not shown). Two guglielmi electrically detachable platinum coils and two hydrosoft helical coils were successfully deployed into the aneurysm . We tried to deploy a microplex coil at the lower neck portion of the aneurysm, but failed due to the wide neck of the aneurysm . The balloon or stent - assisted technique was not used even though there was a relatively large orifice because of the risk of thromboembolism and obstruction of the parent arteries during the procedure . Eventually, only the dome and waist of the aneurysm were packed completely (fig . After embolization, we closely monitored the patient's neurological condition in the intensive care unit . The first follow - up angiograms 6 months after treatment did not show aneurysm recanalization . Atherosclerosis and aortic inflammation are known to be the most common causes of bilateral carotid artery occlusion without moyamoya disease.17) atherosclerosis can occur at the point of ica bifurcation, leading to chronic and progressive arterial stenosis, whereas aortic inflammation is likely to be involved with the aortic arch and its branches, resulting in bilateral carotid artery occlusion . Generally, an occlusion at the beginning part of the ica is likely to be caused by atherosclerosis, while the occlusion of the bilateral common carotid artery is likely aortitis.17) according to the study by aburahma and copeland,1) all patients with atherosclerotic bilateral ica occlusion had a history of cerebral ischemic attack such as transient ischemic attack (tia), amaurosis fugax, or stroke even though collateral circulation was present, and they all needed surgical interventions such as endarterectomy, carotid - subclavian bypass or medical treatments using warfarin or aspirin . By contrast, definite cases of moyamoya disease are diagnosed in patients with bilateral stenosis or occlusion that occurs at the terminal portion of the ica together with an abnormal vascular network at the base of the brain, as is shown by cerebral angiography . Thus, moyamoya syndromes that have underlying diseases such as cerebrovascular disease with atherosclerosis, autoimmune disease, meningitis, brain neoplasm, down's syndrome, neurofibromatosis, head trauma or irradiation to the head, as well as other conditions, should be excluded.9) meanwhile, our case showed complete occlusion at the bilateral proximal portion of the internal carotid arteries above the carotid bulbs with abnormal vascular networks from the posterior circulation without basal moyamoya vessels . The patient did not have any underlying diseases such as cerebral infection, trauma and aortic inflammation that may lead to stenosis or occlusion of the ica . Although he had no past history of tia, he had two risk factors for atherosclerosis which were hypertension and smoking . No abnormal findings that may imply fibromuscular dysplasia were seen on the vertebral and abdominal angiograms . These findings are not characteristics of moyamoya disease or moyamoya syndrome, but suggest idiopathic or atherosclerotic bilateral carotid artery occlusion . The basilar tip aneurysm associated with bilateral carotid artery occlusion or moyamoya disease is well known, life - threatening complication that causes sah . To the best of our knowledge, there have been only seven cases of bilateral carotid artery occlusion with concurrent basilar tip aneurysm without moyamoya disease in the literature.5)7)8)11)12)17)18) the causes of carotid artery occlusion included aortic inflammation in two cases and atherosclerosis in five cases.5)7)8)11)12)17)18) of them, 5 cases had sah.8)11)12)17)18) it has been suggested that increased blood flow through the vertebrobasilar system, which is the major source of collateral circulation in bilateral carotid artery occlusion, intensifies the hemodynamic stress on the arterial walls and this may result in the formation of saccular aneurysms.13) although the formation of anastomosis from the external carotid artery may alleviate the hemodynamic burden on the posterior circulation, the effect is limited and local blood pressure still rises which leads to the risk of basilar artery aneurysms.17) conservative management for these aneurysms can lead to dismal results,11)18) therefore quick, aggressive treatments should be considered . However, direct clipping is difficult and dangerous due to extensive collateral channels,2)14) stiffness of the carotid artery,10)16) and high internal blood pressure.7) moreover, the fact that the basilar artery is the sole source of blood for most of the brain precludes prolonged temporary clipping to aid in safe dissection around the aneurysm.13) thus, these aneurysms have been recently treated with endovascular procedures using soft platinum coils . There have been several reports have showing excellent results of endovascular embolization for the basilar tip aneurysms in patients with moyamoya disease and bilateral ica occlusion.4)6)7)10)12)17) of course, if increased blood burden through the vertebrobasilar system is not alleviated, the aneurysm will probably regrow . Thus, in our case, regular follow - up angiograms are essential and additional embolization with extra - intracranial revascularization may be needed even though there was no perfusion defect seen on the perfusion ct scans . Proximal bilateral ica occlusion is rare but often shows similar clinical features with moyamoya disease such as abnormal collateral vascular networks from posterior circulation and accompanying saccular aneurysms . Differential diagnosis between idiopathic and atherosclerotic occlusion may be needed for further cardiovascular work - up . The basilar tip aneurysm associated with proximal bilateral ica occlusion is more rare and dangerous, but curable disease . Endovascular embolization for such aneurysms is safe and useful even without the assistance of balloon or stent.
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Merkel cell carcinoma, originally described by toker in 1972, is a rare cutaneous malignancy of unknown etiology most commonly seen in the elderly caucasian population . Incidence has been reported at 0.15 cases per 100,000 in 1986 and 0.44 cases per 100,000 in 2001, with increasing incidence attributed to longer average lifespan and advances in diagnostic technology . Mcc is more common in people of advanced age with history of malignancy, immunosuppression, or significant ultraviolet exposure . Recently, infection with the merkel cell polyomavirus has been shown to be a significant risk factor as well [35]. Patients typically present with a firm, painless purplish nodule on the face or upper extremities . Staging is based upon the traditional tnm (tumor - node metastasis) classification system with prognostic data showing decreased overall survival with increasing stage of disease [6, 7]. Oncologic surgery within the head and neck presents unique problems, especially when treating merkel cell carcinoma . Because of the rarity of mcc, there is limited clinical data to guide management . Currently, there are limited clinical guidelines for mcc; however most surgeon clinicians endorse multimodal therapy with wide local excision of the primary tumor and definitive treatment of any clinically significant nodal disease, either with lymphadenectomy or radiation therapy . The role of chemotherapy is unclear, traditionally reserved for diffusely metastatic and/or recurrent disease . Novel targeted therapies are currently being developed and will be discussed elsewhere in this special issue on mcc . . This paper will describe the management options for advanced merkel cell carcinoma as well as the principles of reconstruction when free tissue transfer is utilized . Surgery is the current standard for locoregional disease and has been shown to confer a survival benefit in mcc [814]. However, controversy exists regarding the necessity for wide surgical margins, which can be problematic in the head and neck region . Data regarding mohs surgery in mcc shows comparable, if not superior, local control when compared to traditional surgical excision . As mcc is predominantly found on the sun exposed areas of the head and neck, any surgical intervention will have aesthetic and functional implications . Mohs micrographic surgery provides a more conservative surgical approach while obtaining negative margins and this conserves local tissues . In addition, it has been reported that traditional surgery for mcc frequently results in unrecognized positive deep margins . With mohs surgery, complete excision is more likely; and local recurrence after reconstruction decreases . In general, lesions on the sun - exposed areas of the face, head and neck, mohs micrographic surgery, while accurate, is not ideal for large extensive lesions requiring general anesthetic for ablative surgery and reconstruction . Large lesions may render mohs surgery impractical due to the length of the procedure or depth of invasion . In these situations it should be noted that the author has used a combination technique with mohs micrographic confirmation of negative cutaneous margins prior to traditional wide local excision of the primary tumor and reconstruction . This allows for rapid excision of the primary lesion with mohs micrographic control of the cutaneous margins, which improves accuracy and minimizes unnecessary extension of soft tissue margins . The downside to this technique is the inability to assess the deep margins via the mohs technique and traditional frozen section must be utilized . Regardless of the surgical technique required for excision of the primary tumor, every effort should be made to obtain negative margins . Large extensive lesions at risk for local recurrence should be considered for postoperative radiotherapy regardless of surgical margin status . There is data suggesting radiotherapy in addition to mohs surgery results in improved locoregional control when compared to mohs surgery alone . Primary radiotherapy may be employed in patients with inoperable tumors or comorbidities significant enough to preclude surgery . However, the majority of patients (60%) go on to have out - of - field metastasis . The determination of appropriate treatment fields for postoperative radiotherapy remains controversial and should be determined by the radiation oncologist after evaluating the patient and operative results . Additional information regarding radiotherapy for mcc will be presented elsewhere in this special issue . At present, the recommendation for management of lymph node disease in mcc depends on clinical presentation . For clinically significant lymph node extension cervical lymphadenectomy or therapeutic radiation therapy is indicated after histological confirmation [13, 14]. There is data suggesting that the size of the primary tumor correlates with the risk of occult disease and that occult disease is unlikely with a primary tumor less than or equal to 1 cm . Sentinel lymph node (sln) biopsy has become a useful tool in attaining a reliable histological indicator of nodal spread and limited data shows decreased recurrence rates where regional management was influenced by sentinel lymph node biopsy . At present, there is insufficient data to determine standardized guidelines for sln or elective lymph node dissection in the clinically negative neck for mcc . For the majority of surgical defects in the region of the head and neck, locoregional reconstruction with skin grafting or local flaps is functionally and aesthetically adequate . A comprehensive review of factors, which influence the reconstructive approach, is beyond the scope of this paper and will be reviewed elsewhere in the special issue . The extent of disease, viability and quality of surrounding tissue, involvement of the adjacent structures, and history of prior surgery or radiation therapy can make locoregional reconstruction less appealing or impossible . Given the importance of negative surgical margins in mcc, the oncologic surgeon must consider the implications of the risk of local recurrence and consider the propensity for multiple synchronous tumors as well as immune system dysfunction in elderly patients with extensive disease . In these situations, conservative surgery may not be possible, resulting in significant ablative defects of the head and neck . In these situations, free tissue transfer can provide large volume, healthy tissue for reconstruction of the surgical defect, with favorable aesthetic and functional outcomes . The increased application of microsurgical reconstruction has resulted in several options for free tissue transfer for soft tissue defects of the head and neck . The anterolateral thigh (alt) flap, latissimus dorsi, rectus abdominis, scapula / parascapular, and radial forearm flap (rfff), flap selection depends on the tissue components of the defect (i.e., skin, muscle, and bone) as well as the location, size, depth, and surrounding tissue color / contour . Other aspects during reconstruction such as facial nerve involvement, availability of donor vessels, and donor site considerations such as peripheral vascular disease may alter the reconstructive plan . When considering mcc specifically, the data shows frequent local recurrence as well as a propensity for vertical invasion with positive deep margins being relatively common . These considerations often result in an extensive soft tissue resection involving underlying fat and muscle . In this case, the flaps mentioned above provide a great deal of bulk, require a straightforward harvest with little donor site morbidly, and provide adequate vascular pedicles to limit the need for vein grafting . For defects in the dura, tensor fascia lata grafting is commonly employed with good results . Donor vessels commonly used are superficial temporal system, facial artery, superior thyroid artery, and transverse cervical system [21, 22]. Donor site selection involves a relatively complicated assessment of the ablative defect, the composition of the defect, and the overall condition of the patient . Options for soft tissue reconstruction will be discussed briefly; however it should be noted that a variety of other techniques (i.e., osteomyocutaneous flaps) may be employed if dictated by the defect . The ability to provide large volumes of well - vascularized composite tissue is the most significant advantage of free tissue transfer techniques, when managing large volume defects related to mcc . The alt flap has become a popular reconstructive option for surgical defects within the head and neck . It is easily harvested via a two - team approach, with low donor site morbidity and provides a large softtissue volume, a long and reliable vascular pedicle . The alt offers the option for dynamic facial nerve reconstruction via motor nerve to the vastus lateralis . The pedicle length limits the necessity for venous grafting and allows microsurgical anastomosis of vessels to occur some distance from the defect [19, 22]. The alt flap has become the author's preferred workhorse flap for defects> 100 sq cm, or in cases where thicker tissue is desired (figure 1). The latissimus dorsi flap has found use predominantly in reconstruction of scalp defects, especially those with exposed calvarium . It is harvested easily, provides a large surface area with excellent muscle volume and thickness . Atrophy of the graft provides results in close contour matching with surrounding skin and soft tissue . In most scenarios, this flap should be deepithelialized and covered with a split thickness skin graft for better aesthetic color, matching, and thickness . O'connell et al . Retrospectively evaluated 65 patients with scalp or lateral temporal bone defects, performing a total of 68 free tissue transfers . Based on their experience, the latissimus muscle - only flap with split - thickness skin graft (stsg) provides a large surface area, adequate bulk, and an aesthetically acceptable result in a large majority of scalp defects . Donor site morbidity is generally well tolerated; however in elderly patients who used stabilizing devices such as canes, walkers, or are wheelchair dependent, the alt may be a more favorable option . For smaller defects requiring thin and pliable tissue, primarily a fasciocutaneous flap is ideally suited to smaller defects with complex contours where it is desirable to avoid excessive bulk . The reliable pedicle length and predictability of harvest make the radial forearm free flap a commonly utilized reconstructive technique for cutaneous defects of the head and neck . In cases with vessel - depleted necks where veins are unavailable, a semifree radial forearm harvest has been described dissecting the cephalic vein proximally and performing a single arterial anastomosis, which is unique to this flap . Donor site morbidity is well tolerated, however generally inferior to the anterior thigh flap [24, 25] (figures 2, 3, and 4). The rectus abdominis flap has been used for head and neck reconstruction for cutaneous malignancies and offers the advantage of well - vascularized muscle and large amounts of soft tissue available for reconstruction . When large amounts of muscles are harvested, donor site morbidity is increased and therefore, it is our opinion that other free tissue donor sites offer several advantages over the rectus abdominis flap in the majority of cases . Muscle sparing perforator style flaps may allow for decreased morbidity and superior control of flap thickness, and the deep inferior epigastria artery - based flaps have been shown to be a valid reliable option for head and neck reconstruction . The scapular / parascapular flap also has excellent contour and color matching with the forehead and scalp and can typically be closed primarily after harvest with very little donor site morbidity [19, 27]. Harvest may be performed with turned supine positioning and large soft tissue flaps may be harvested . Some surgeons prefer the color and thickness of the scapular / parascapular flap for head and neck reconstruction . In addition, the availability of osseous harvest makes the subscapular system the most versatile flap for complex head and neck ablative defects . Merkel cell carcinoma presents frequently on the face and the propensity for vertical invasion often puts the facial nerve at risk . Therefore, there is potential for facial nerve injury secondary to extension of the primary tumor and/or surgical excision for adequate margins (figure 4). Facial nerve injury can result in lifelong facial asymmetry with profound physiological and psychological consequences, especially in the context of a surgical defect . A discussion of facial nerve reanimation is beyond the scope of this paper; however reconstructive flap selection in free tissue transfer may allow dynamic (latissimus, alt) or static (alt, radial forearm / palmaris) reconstructive procedures to be performed simultaneously . Selection of free tissue donor sites should consider the desired facial nerve reanimation strategy in order to minimize additional donor sites . Reconstruction after wide excision of mcc offers several unique challenges including a propensity for elderly patients with poor tissue quality and decreased immune function, large defects and a high probability of disease recurrence, and the risks inherent to adjuvant radiotherapy . For this reason, most reconstructive surgeons favor free tissue transfer as a modality for providing healthy, uninvolved tissue with an acceptable aesthetic outcome . There is clearly a need for higher quality of data in the area, as significant questions remain for the treatment for merkel cell cancer, as well as the reconstructive methods utilized after ablative surgery . Recently, data has been published suggesting that wide surgical margins during tumor resection may not impact overall survival in patients receiving adjuvant radiotherapy . This is a crucial question for the reconstructive surgeon as it may impact defect size and the probability of local recurrence . Additional data on radiotherapy and targeted chemotherapy for mcc may play a role in deciding the timing for reconstruction, as well as the selected technique . The decision on surgical approach by the reconstructive surgeon should be based on the individual patient and should take into account the details of the clinical scenario in addition to the location and size of the defect . Current free tissue transfer techniques allow the reconstructive surgeon to manage advanced mcc with acceptable functional and aesthetic results, while minimizing morbidity.
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Ketamine has been used clinically since the 1960s as a general anesthetic.1 a decade after its approval by the us food and drug administration, researchers showed that ketamine reduces the excitatory response to n - methyl - d - aspartic acid (nmda) in central neurons, thus revealing its nmda receptor antagonism properties2 and advancing our understanding of the pharmacology of ketamine . Currently, clinicians use ketamine as a general anesthetic less frequently, given its emergence psychotomimetic effects and findings of neurotoxic effects shown in the developing brain of animals.3 conversely, over the past decade, researchers have explored the effect of subanesthetic doses of ketamine as an antidepressant4 and as an analgesic.5,6 the use of subanesthetic ketamine as an analgesic is supported by evidence, from both animal and human studies, implicating activation of nmda receptors in the pathobiology of nociceptive, inflammatory, and neuropathic pain and in central sensitization.7 in addition, activation of nmda receptors has also been shown in animals8 and in human experimental studies9 to play a role in settings where acute or chronic use of opioids is associated with tolerance or opioid - induced hyperalgesia . Therefore, based on these preclinical findings, ketamine has been examined clinically as an adjuvant to opioids for the treatment of acute and chronic pain and in settings of opioid - induced hyperalgesia.6,10 however, among clinical studies, there has been a great variability in ketamine doses and duration of administration . Additionally, the beneficial effects of ketamine have not been consistently reported, and some groups have been unable to show opioid - sparing effects of ketamine in clinical settings associated with opioid - induced hyperalgesia.11,12 similarly, in chronic pain, there are no definitive data supporting the use of ketamine for the treatment of chronic regional pain syndrome (crps) or other types of chronic pain.13 further, in critical reviews of available trials, researchers can only conclude that there might be a beneficial role for ketamine as an adjuvant to opioids to treat pain in the postoperative period, cancer pain, and chronic pain.9,12,14,15 consequently, as outlined in clinical guidelines, the evidence to support the use of ketamine for the treatment of acute and chronic pain is at best moderate.16,17 however, despite lack of conclusive clinical evidence to support its use, clinicians from various disciplines in many countries continue to use ketamine as an adjuvant to opioids for acute and chronic pain management.1821 some argue that in the setting of limited effective pharmacologic options, one might be left with no alternative other than less studied strategies such as ketamine infusions . Our institution has used subanesthetic ketamine (defined as not> 1 mg / kg / h) to treat acute and chronic pain in children, adolescents, and young adults since 2006 . We have previously reported our experience using ketamine for the treatment of cancer pain, postoperative, and chronic pain.5,11 in this study, we sought to examine the use of subanesthetic ketamine in a large number of patients admitted to a tertiary pediatric hospital whose pain proved unresponsive to routine pharmacologic interventions . Specifically, we sought to determine the overall effect of ketamine on pain scores and opioid intake, to identify factors associated with larger or smaller effects and to examine the frequency with which ketamine was associated with clinically meaningful reductions in pain scores and opioid intake . The children s national health system institutional review board approved this longitudinal cohort study and a waiver for written informed consent in 2014 (pro00004569). Requirement for patient written consent to review their records was waived as this was a minimal risk study and the data collected were devoid of patient identifiers . Patients who had received subanesthetic doses of ketamine for pain management were eligible for inclusion in this study . We reviewed a list of all patients (obtained from the hospital pharmacy records) who were admitted between january 2006 and april 2014 and had received an order for continuous infusion of intravenous ketamine . Patients who lacked a pain medicine consult, underwent a spinal fusion procedure (those patients were part of a previously reported investigational study11), had incomplete record of pain scores and/or opioid intake, or received ketamine only for the purpose of sedation were excluded . Data collected included sex, age, race, ketamine infusion duration, pain duration (acute vs chronic), pain diagnoses (vaso - occlusive episodes [voe], visceral, crps, headache, or other [mucositis, diabetic neuropathy, neurofibromatosis, and postural orthostatic tachycardia syndrome]), primary clinical diagnosis (accidental trauma, surgical trauma, and postoperative pain [patients who had a planned surgical procedure during hospitalization], functional gastrointestinal disorder [chronic and/or recurrent gastrointestinal symptoms with no identified etiology], inflammatory disease [pancreatitis or crohn s disease], sickle cell disease, malignancy, or other), and primary pain location . Patients with pain in more than two locations, we retrieved documentation of hemodynamic changes that warranted treatment with vasoactive agents, as well as reports of psychotropic effects, nausea, vomiting, and changes in sleep pattern . We then calculated average daily pain scores before (day prior to ketamine administration) and after (24 h, 48 h, and on the day after discontinuation) ketamine infusion . Pain scores were measured using the numeric rating scale (nrs), the wong - baker faces, or the face, legs, activity, cry, and consolability (flacc), as appropriate . At the times indicated earlier, we also collected total daily opioid intake and then calculated the daily oral morphine - equivalent (per body weight in kilograms) intake using the opioid conversion tool available at www.globalrph.com/narcoticonv.htm.22 the primary outcome was change in average pain scores from baseline to postketamine infusion . Secondary outcomes included changes in oral morphine - equivalent intake from baseline measurements to postketamine infusion and proportion of ketamine infusions that resulted in clinically meaningful reductions in pain scores and opioid intake . We defined clinically meaningful change in pain scores or opioid intake as a 20% reduction in pain scores and in morphine - equivalent intake from baseline to the day after ketamine discontinuation (day after ketamine - baseline / baseline 100). Such a decline in pain score (20%) is larger than the 12.5% decrease in pain score suggested by others as being minimally clinically significant for adolescents with chronic pain.23 at our institution, ketamine prescribed by the pain medicine division can be administered on regular inpatient care units where oxygen delivery set - up, suction, and resuscitation equipment are available . Trained nurses administer ketamine infusions using a locked infusion pump and patients are maintained on a cardiorespiratory monitor (heart and respiratory rate, blood pressure, and pulse oximeter). Criteria to receive subanesthetic doses of ketamine infusions include inadequate analgesia with standard pain therapy, opioid tolerance associated with undesirable side effects, opioid - induced hyperalgesia, neuropathic pain, postoperative pain in patients in whom opioid use was undesirable, or an opioid - sparing effect was needed for clinical reasons . Exclusion criteria for ketamine administration include history of psychosis, intracranial hypertension, severe labile hypertension, or poorly controlled cardiac arrhythmia, hypersensitivity to ketamine, and age <3 months . In our institution, ketamine infusions order sets are available, and the suggested starting doses are as follows: 1) for opioid naive patients, ketamine is started at 0.050.4 mg / kg / h; 2) for opioid tolerant patients (as documented by the pain service), 0.051 mg / kg / h; and 3) for patients with documented opioid - induced hyperalgesia, 1 mg / kg / h . During infusions, ketamine doses could be titrated according to its indication and pain scores up to 1 mg / kg / h as allowed by the board of nursing . The distributions of raw pain scores, daily opioid intake, and changes over time were tested for normality . Because the data were skewed, the wilcoxon signed - rank test was used to test for statistical significance of within - individuals changes between baseline and posttreatment outcome measurements . Box plots were used to examine how the individual - level changes (between baseline and the day after ketamine discontinuation) in raw pain scores and daily opioid intake were distributed across the subgroups of the eight patient characteristics (age, sex, race, duration of pain, clinical diagnosis, type of pain, location of pain, and duration of ketamine infusion), and the distributions were tested for normality . Because the data were skewed for certain characteristics, nonparametric tests were used for all statistical analyses . The kruskal wallis test was used to compare the distribution of values across the subgroups of each patient characteristic separately . When an overall difference was detected, dunn s test (with a bonferroni adjustment for multiple comparisons) was then used to compare all possible pairs of subgroups and identify any statistically significant detectable differences . The proportion of patients (95% confidence interval [ci]) who achieved clinically meaningful reductions in pain and daily opioid intake were graphed in order to visually compare how the proportions varied across the subgroups of all eight patient characteristics . Fisher s exact test was used to examine whether the proportions varied across the subgroups of sex, race, duration of pain, clinical diagnosis, type of pain, or location of pain . A nonparametric test for trend was used to examine whether the proportion of patients with a clinically meaningful reduction in pain scores and opioid intake systematically declined across ordered age groups or by the duration of ketamine infusion . Stata 14.1/ic (statacorp llc, college station, tx, usa) was used to conduct all analyses . P - values <0.05 were considered statistically significant except when bonferroni s correction for multiple comparisons was applied . In those instances, the children s national health system institutional review board approved this longitudinal cohort study and a waiver for written informed consent in 2014 (pro00004569). Requirement for patient written consent to review their records was waived as this was a minimal risk study and the data collected were devoid of patient identifiers . Patients who had received subanesthetic doses of ketamine for pain management were eligible for inclusion in this study . We reviewed a list of all patients (obtained from the hospital pharmacy records) who were admitted between january 2006 and april 2014 and had received an order for continuous infusion of intravenous ketamine . Patients who lacked a pain medicine consult, underwent a spinal fusion procedure (those patients were part of a previously reported investigational study11), had incomplete record of pain scores and/or opioid intake, or received ketamine only for the purpose of sedation were excluded . Data collected included sex, age, race, ketamine infusion duration, pain duration (acute vs chronic), pain diagnoses (vaso - occlusive episodes [voe], visceral, crps, headache, or other [mucositis, diabetic neuropathy, neurofibromatosis, and postural orthostatic tachycardia syndrome]), primary clinical diagnosis (accidental trauma, surgical trauma, and postoperative pain [patients who had a planned surgical procedure during hospitalization], functional gastrointestinal disorder [chronic and/or recurrent gastrointestinal symptoms with no identified etiology], inflammatory disease [pancreatitis or crohn s disease], sickle cell disease, malignancy, or other), and primary pain location . Patients with pain in more than two locations, we retrieved documentation of hemodynamic changes that warranted treatment with vasoactive agents, as well as reports of psychotropic effects, nausea, vomiting, and changes in sleep pattern . We then calculated average daily pain scores before (day prior to ketamine administration) and after (24 h, 48 h, and on the day after discontinuation) ketamine infusion . Pain scores were measured using the numeric rating scale (nrs), the wong - baker faces, or the face, legs, activity, cry, and consolability (flacc), as appropriate . At the times indicated earlier, we also collected total daily opioid intake and then calculated the daily oral morphine - equivalent (per body weight in kilograms) intake using the opioid conversion tool available at www.globalrph.com/narcoticonv.htm.22 secondary outcomes included changes in oral morphine - equivalent intake from baseline measurements to postketamine infusion and proportion of ketamine infusions that resulted in clinically meaningful reductions in pain scores and opioid intake . We defined clinically meaningful change in pain scores or opioid intake as a 20% reduction in pain scores and in morphine - equivalent intake from baseline to the day after ketamine discontinuation (day after ketamine - baseline / baseline 100). Such a decline in pain score (20%) is larger than the 12.5% decrease in pain score suggested by others as being minimally clinically significant for adolescents with chronic pain.23 at our institution, ketamine prescribed by the pain medicine division can be administered on regular inpatient care units where oxygen delivery set - up, suction, and resuscitation equipment are available . Trained nurses administer ketamine infusions using a locked infusion pump and patients are maintained on a cardiorespiratory monitor (heart and respiratory rate, blood pressure, and pulse oximeter). Criteria to receive subanesthetic doses of ketamine infusions include inadequate analgesia with standard pain therapy, opioid tolerance associated with undesirable side effects, opioid - induced hyperalgesia, neuropathic pain, postoperative pain in patients in whom opioid use was undesirable, or an opioid - sparing effect was needed for clinical reasons . Exclusion criteria for ketamine administration include history of psychosis, intracranial hypertension, severe labile hypertension, or poorly controlled cardiac arrhythmia, hypersensitivity to ketamine, and age <3 months . In our institution, ketamine infusions order sets are available, and the suggested starting doses are as follows: 1) for opioid naive patients, ketamine is started at 0.050.4 mg / kg / h; 2) for opioid tolerant patients (as documented by the pain service), 0.051 mg / kg / h; and 3) for patients with documented opioid - induced hyperalgesia, 1 mg / kg / h . During infusions, ketamine doses could be titrated according to its indication and pain scores up to 1 mg / kg / h as allowed by the board of nursing . The distributions of raw pain scores, daily opioid intake, and changes over time were tested for normality . Because the data were skewed, the wilcoxon signed - rank test was used to test for statistical significance of within - individuals changes between baseline and posttreatment outcome measurements . Box plots were used to examine how the individual - level changes (between baseline and the day after ketamine discontinuation) in raw pain scores and daily opioid intake were distributed across the subgroups of the eight patient characteristics (age, sex, race, duration of pain, clinical diagnosis, type of pain, location of pain, and duration of ketamine infusion), and the distributions were tested for normality . Because the data were skewed for certain characteristics, nonparametric tests were used for all statistical analyses . The kruskal wallis test was used to compare the distribution of values across the subgroups of each patient characteristic separately . When an overall difference was detected, dunn s test (with a bonferroni adjustment for multiple comparisons) was then used to compare all possible pairs of subgroups and identify any statistically significant detectable differences . The proportion of patients (95% confidence interval [ci]) who achieved clinically meaningful reductions in pain and daily opioid intake were graphed in order to visually compare how the proportions varied across the subgroups of all eight patient characteristics . Fisher s exact test was used to examine whether the proportions varied across the subgroups of sex, race, duration of pain, clinical diagnosis, type of pain, or location of pain . A nonparametric test for trend was used to examine whether the proportion of patients with a clinically meaningful reduction in pain scores and opioid intake systematically declined across ordered age groups or by the duration of ketamine infusion . Stata 14.1/ic (statacorp llc, college station, tx, usa) was used to conduct all analyses . P - values <0.05 were considered statistically significant except when bonferroni s correction for multiple comparisons was applied . In those instances, we reviewed 1542 records, containing orders for subanesthetic ketamine administration (figure 1). Records containing ketamine orders not written by the pain medicine division were incomplete or were from patients who had undergone spinal fusion (ketamine was administered as part of a postoperative pain management and previously reported11) were excluded . Two hundred and fifty - one patients and 441 infusions, which occurred between january 2006 and april 2014, met the study eligibility criteria . Of the 441 remaining records, 57 were excluded as pain scores, and/or opioid intake data were missing during the first or second day of ketamine administration or for the 24 h after discontinuation of ketamine . Records from 24 ketamine infusions were eliminated because they consisted of a second ketamine infusion during a single hospital admission . The final study cohort included data from 230 different patients who, during 360 separate hospital admissions, received subanesthetic ketamine infusions for pain therapy . Of those, 180 patients were admitted once, while the other 50 were each admitted between two and ten times . For the purpose of this study, over the 360 ketamine infusions, most patients were from 12 to 18 years of age (58%), 6 to 11 years (17%), 19 to 21 years (14%), 3 to 5 years (5%),> 22 years (4%), and 1.1 to 2.9 years (3%). Seventy - eight percent of infusions (280) were administered to patients with acute pain and 22% to patients with chronic pain . Additionally, most ketamine infusions (50%) were administered to patients with sickle cell disease who had been admitted for voe, whereas 17% who had postoperative pain after surgical trauma and 13% had malignancy - associated pain (table 2). Most patients had generalized pain (25%), followed by abdominal (22%), lower extremity (21%), and chest (15%) pain . We collected average pain scores on the day prior to initiation of ketamine and on the first day of ketamine infusion . In the hospital admissions (n=331 of 441) where both values were originally available in the electronic medical record, the average pain score on the day prior to ketamine initiation (6.85 [95% ci: 6.587.11]) and on the first day of ketamine infusion were similar (6.84 [95% ci: 6.597.08]), p=0.499 . Given that in all infusions, the average pain score values were available on the first day of infusion, we considered that as the baseline pain score for the final study cohort of 360 hospital admissions . Overall, ketamine infusions were associated with significant reductions in average pain scores from the first day of infusion (6.65 [95% ci: 6.396.91]) to the day after discontinuation of ketamine (4.38 [95% ci: 4.064.69]), p<0.001 . The effect of ketamine infusions on raw pain scores (within - individual change) was similar comparing all age groups (p=0.842), males and females (p=0.065), all races (p=0.721), acute and chronic pain (p=0.460), and pain types (p=0.894), data not shown . In contrast, the effect of ketamine infusions on pain scores varied according to clinical diagnosis (p=0.01), infusion duration (p=0.004), and pain location (p=0.004, figure 2). Specifically, greater median reductions in pain scores were observed in patients with malignancy - associated pain and patients with inflammatory disease (pancreatitis and crohn s disease), whereas the lowest were in patients with functional gastrointestinal disorder and other (cystic fibrosis, diabetes mellitus, neurofribromatosis 1, postural orthostatic tachycardia syndrome, and two cases simply coded as other, p=0.011 for overall difference, figure 2a). Regarding infusion durations, longer ketamine infusions yielded greater median reductions in pain scores (p=0.004, figure 2b). Further, patients with back (2.57 [4.75, 1.56], median [25, 75 percentiles respectively]), abdominal (1.91 [3.88, 0.6]), and generalized (1.87 [3.43, 0.75]) pain had the greatest reductions, whereas patients with chest pain (0.5 [2.37, 0.33]) had the lowest reductions in pain scores (p=0.004 for overall difference). Regarding opioid intake, ketamine infusions were associated with significant decreases in overall average oral morphine - equivalent intake from the first day of infusion (2.74 mg / kg / d [2.473.00], mean [95% ci]) to the day after discontinuation of ketamine (2.06 mg / kg / d [1.812.31]), p<0.001 . There was no impact of age, sex, race, pain duration (acute vs chronic), or pain location or type on the effect of ketamine on oral morphine - equivalent intake (all p0.322). However, the ketamine effect on opioid intake varied according to clinical diagnosis (p=0.030, figure 2c) and duration of infusion (p=0.022, figure 2d). Specifically, greater median reductions in opioid intake were observed in patients with malignancy - associated pain and patients with sickle cell disease, whereas lesser reductions were observed in patients with accidental trauma and postoperative pain after surgical trauma (p=0.030 for overall difference, figure 2c). Regarding ketamine infusion duration, similar to the observation of reduction in pain scores, longer ketamine infusions yielded greater reductions in oral morphine - equivalent intake (p=0.022, figure 2d). During ketamine administration, there were no records of hallucinations, changes in sleep pattern, or hemodynamic changes or arrhythmias requiring therapy in any patient . We then examined the proportion of ketamine infusions that yielded clinically meaningful changes in pain scores and opioid intake, here defined as a 20% reduction in pain scores and a 20% reduction in morphine - equivalent intake after discontinuation of the infusion compared to baseline levels, respectively . Overall, there was a 20% reduction in pain scores in 58% of all ketamine infusions . The proportion of infusions that yielded a 20% or greater reduction in pain scores varied according to clinical diagnosis (p=0.003), pain location (p=0.012), age (p=0.039), and duration of infusion (p=0.017, figure 3). For example, the highest proportion of infusions associated with clinically meaningful reduction in pain scores were observed among infusions administered to cancer patients and patients with inflammatory diseases and the lowest among patients with functional gastrointestinal disorders (figure 3a). There was a trend for higher frequency of meaningful reduction in pains scores in males (63%) than in females (54%), p=0.056 . Conversely, the frequency of meaningful reduction in pain scores did not vary with the type (p=0.391) and duration of pain (p=0.224) or race (p=0.268). Regarding the frequency of a meaningful reduction in opioid intake, in 52% of ketamine infusions the frequency of infusions associated with 20% reduction in opioid intake varied according to duration of ketamine infusion (p=0.030), with longer durations infusions being more likely to result in a meaningful reduction in opioid intake (data not shown). Conversely, the frequency of a meaningful reduction in opioid intake did not vary with the clinical diagnosis, type of pain, duration of pain, location of pain, age, sex, or race . We reviewed 1542 records, containing orders for subanesthetic ketamine administration (figure 1). Records containing ketamine orders not written by the pain medicine division were incomplete or were from patients who had undergone spinal fusion (ketamine was administered as part of a postoperative pain management and previously reported11) were excluded . Two hundred and fifty - one patients and 441 infusions, which occurred between january 2006 and april 2014, met the study eligibility criteria . Of the 441 remaining records, 57 were excluded as pain scores, and/or opioid intake data were missing during the first or second day of ketamine administration or for the 24 h after discontinuation of ketamine . Records from 24 ketamine infusions were eliminated because they consisted of a second ketamine infusion during a single hospital admission . The final study cohort included data from 230 different patients who, during 360 separate hospital admissions, received subanesthetic ketamine infusions for pain therapy . Of those, 180 patients were admitted once, while the other 50 were each admitted between two and ten times . For the purpose of this study, the median age was 14 years (interquartile range: 1017 years). Over the 360 ketamine infusions, most patients were from 12 to 18 years of age (58%), 6 to 11 years (17%), 19 to 21 years (14%), 3 to 5 years (5%),> 22 years (4%), and 1.1 to 2.9 years (3%). Seventy - eight percent of infusions (280) were administered to patients with acute pain and 22% to patients with chronic pain . Additionally, most ketamine infusions (50%) were administered to patients with sickle cell disease who had been admitted for voe, whereas 17% who had postoperative pain after surgical trauma and 13% had malignancy - associated pain (table 2). Most patients had generalized pain (25%), followed by abdominal (22%), lower extremity (21%), and chest (15%) pain . We collected average pain scores on the day prior to initiation of ketamine and on the first day of ketamine infusion . In the hospital admissions (n=331 of 441) where both values were originally available in the electronic medical record, the average pain score on the day prior to ketamine initiation (6.85 [95% ci: 6.587.11]) and on the first day of ketamine infusion were similar (6.84 [95% ci: 6.597.08]), p=0.499 . Given that in all infusions, the average pain score values were available on the first day of infusion, we considered that as the baseline pain score for the final study cohort of 360 hospital admissions . Overall, ketamine infusions were associated with significant reductions in average pain scores from the first day of infusion (6.65 [95% ci: 6.396.91]) to the day after discontinuation of ketamine (4.38 [95% ci: 4.064.69]), p<0.001 . The effect of ketamine infusions on raw pain scores (within - individual change) was similar comparing all age groups (p=0.842), males and females (p=0.065), all races (p=0.721), acute and chronic pain (p=0.460), and pain types (p=0.894), data not shown . In contrast, the effect of ketamine infusions on pain scores varied according to clinical diagnosis (p=0.01), infusion duration (p=0.004), and pain location (p=0.004, figure 2). Specifically, greater median reductions in pain scores were observed in patients with malignancy - associated pain and patients with inflammatory disease (pancreatitis and crohn s disease), whereas the lowest were in patients with functional gastrointestinal disorder and other (cystic fibrosis, diabetes mellitus, neurofribromatosis 1, postural orthostatic tachycardia syndrome, and two cases simply coded as other, p=0.011 for overall difference, figure 2a). Regarding infusion durations, longer ketamine infusions yielded greater median reductions in pain scores (p=0.004, figure 2b). Further, patients with back (2.57 [4.75, 1.56], median [25, 75 percentiles respectively]), abdominal (1.91 [3.88, 0.6]), and generalized (1.87 [3.43, 0.75]) pain had the greatest reductions, whereas patients with chest pain (0.5 [2.37, 0.33]) had the lowest reductions in pain scores (p=0.004 for overall difference). Regarding opioid intake, ketamine infusions were associated with significant decreases in overall average oral morphine - equivalent intake from the first day of infusion (2.74 mg / kg / d [2.473.00], mean [95% ci]) to the day after discontinuation of ketamine (2.06 mg / kg / d [1.812.31]), p<0.001 . There was no impact of age, sex, race, pain duration (acute vs chronic), or pain location or type on the effect of ketamine on oral morphine - equivalent intake (all p0.322). However, the ketamine effect on opioid intake varied according to clinical diagnosis (p=0.030, figure 2c) and duration of infusion (p=0.022, figure 2d). Specifically, greater median reductions in opioid intake were observed in patients with malignancy - associated pain and patients with sickle cell disease, whereas lesser reductions were observed in patients with accidental trauma and postoperative pain after surgical trauma (p=0.030 for overall difference, figure 2c). Regarding ketamine infusion duration, similar to the observation of reduction in pain scores, longer ketamine infusions yielded greater reductions in oral morphine - equivalent intake (p=0.022, figure 2d). During ketamine administration, there were no records of hallucinations, changes in sleep pattern, or hemodynamic changes or arrhythmias requiring therapy in any patient . We then examined the proportion of ketamine infusions that yielded clinically meaningful changes in pain scores and opioid intake, here defined as a 20% reduction in pain scores and a 20% reduction in morphine - equivalent intake after discontinuation of the infusion compared to baseline levels, respectively . Overall, there was a 20% reduction in pain scores in 58% of all ketamine infusions . The proportion of infusions that yielded a 20% or greater reduction in pain scores varied according to clinical diagnosis (p=0.003), pain location (p=0.012), age (p=0.039), and duration of infusion (p=0.017, figure 3). For example, the highest proportion of infusions associated with clinically meaningful reduction in pain scores were observed among infusions administered to cancer patients and patients with inflammatory diseases and the lowest among patients with functional gastrointestinal disorders (figure 3a). There was a trend for higher frequency of meaningful reduction in pains scores in males (63%) than in females (54%), p=0.056 . Conversely, the frequency of meaningful reduction in pain scores did not vary with the type (p=0.391) and duration of pain (p=0.224) or race (p=0.268). Regarding the frequency of a meaningful reduction in opioid intake, in 52% of ketamine infusions the frequency of infusions associated with 20% reduction in opioid intake varied according to duration of ketamine infusion (p=0.030), with longer durations infusions being more likely to result in a meaningful reduction in opioid intake (data not shown). Conversely, the frequency of a meaningful reduction in opioid intake did not vary with the clinical diagnosis, type of pain, duration of pain, location of pain, age, sex, or race . In this cohort study involving a large number of children, adolescents, and young adults, we examined the effects of subanesthetic ketamine infusions on pain intensity and opioid intake . We found that continuous subanesthetic ketamine infusions administered to children, adolescents, and young adults with inadequate pain control in a regular inpatient care unit setting were safe and not associated with significant psychotropic effects or hemodynamic side effects that warranted treatment . We do acknowledge that increases in blood pressure could occur during administration of ketamine; however, we recorded only instances where changes in blood pressure warranted treatment with vasoactive drugs . Further, while there were no records of hallucinations, it is possible that the presence of hallucinations in young children could have manifested as dysphoria and those were not recorded . Nevertheless, ketamine was associated with significant decreases both in pain scores and opioid intake and these effects varied according to clinical diagnosis, infusion duration, and pain location . The greatest reductions in pain scores were observed in patients with cancer pain and patients with inflammatory processes including pancreatitis and crohn s disease, whereas patients with functional gastrointestinal disorder, diabetes mellitus, neurofibromatosis 1, and postural orthostatic tachycardia syndrome had less reduction in pain scores . We also found that ketamine infusions in patients with cancer pain, inflammatory diseases, and postoperative pain were associated with a greater (over 75% of infusions) frequency of meaningful reduction in pain scores (here defined as a 20% reduction). Therefore, this study is informative in that its findings can be used to help design much - needed randomized trials to evaluate the effect of ketamine in the treatment of pain in children, adolescents, and young adults with poorly controlled pain . Over the past few years, the use of ketamine for the treatment of cancer pain has been the focus of significant debate.15,24,25 the discussion ensued after publication of a randomized placebo - controlled clinical trial enrolling 185 adult participants with cancer pain, who received ketamine or placebo delivered subcutaneously.26 that trial showed that ketamine, administered over 5 days in a dose - escalating regimen, had no clinical benefit compared to placebo and was associated with increased adverse events.26 however, researchers argue that the trial used a subcutaneous regimen not commonly utilized, the ketamine doses were escalated rapidly, and the study population was very heterogeneous, all of which are potential limitations of that trial.25 therefore, researchers argue that those issues should tailor the interpretation of the findings26 and those negative results should not discard the potential role of ketamine in patients with cancer pain.25 after publication of those negative results, a survey of clinicians demonstrated that the practice of using ketamine was continued by 30% of the respondents24 and the use of ketamine to treat cancer pain and other types of pain by many specialists has continued.19,24 on balance, one must recognize that the existing data on the use of ketamine in children with cancer pain are very limited, most reports consist of small case series, and randomized clinical trials of ketamine to treat cancer pain in children are lacking . Nevertheless, we have sided with those who argue that, while clinical trials are needed, the practice of using ketamine in children with cancer pain might be one to be adopted when other options are unavailable . Concerning acute postoperative pain, we found in the present study that the opioid - sparing effect of ketamine was minimal . This finding is consistent with our results from a previous randomized placebo - controlled trial, where ketamine administered in the intraoperative and postoperative periods did not yield an opioid - sparing effect in adolescents with scoliosis undergoing spinal fusion.11 interestingly, a more recent trial and a new meta - analysis12 also showed lack of beneficial effect of ketamine in postoperative pain.27 it is important to note that one limitation of the most recent study in adolescents undergoing spinal fusion27 as well as ours11 is that neither study examined the effect of ketamine on chronic postsurgery pain . Nevertheless, our findings from the present study are discrepant to those showing that low - dose ketamine infusion during the perioperative period yields opioid - sparing effects in adults undergoing major surgeries.28 while we acknowledge that, in adults, there are data to support the use of ketamine in the perioperative period, in our institution, we no longer routinely use ketamine to treat postoperative pain in our pediatric patients . One must acknowledge the limitations of this study, which call for caution in interpreting its results . First, we report on the use of ketamine in a heterogeneous population of children, adolescents, and young adults and lack a control group . Further, while there was consistency regarding the ketamine administration regimen and indications for its use, timing of administration, and titration of opioids and ketamine doses were left to the discretion of the pain medicine attending . Additionally, for the purpose of this study, we regarded ketamine infusions administered to the same patients as independent infusions and we did not account for the potential confounding effect of repeated measures by repeatedly administering ketamine to some patients . In conclusion, despite its limitations, this study suggests that in an inpatient setting, the treatment of pain in children, adolescents, and young adults with subanesthetic doses of ketamine is feasible and safe in regular patient care units . The study is also informative in that it suggests that ketamine might have a differential beneficial effect in patients with certain pain syndromes . Additionally, this study raises a number of hypotheses to be tested in future clinical trials to more definitively determine which patients would benefit from this therapeutic modality . Finally, our findings can be used to determine sample and effect sizes for future randomized trials to evaluate the role of ketamine for the treatment of pain in children, adolescents, and young adults.
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Nuclear domain 10 (also called kremer bodies or pods) was first observed almost 50 years ago by electron microscopy as nuclear dense granular bodies1, 2 . These punctuate structures are now dubbed pml nuclear bodies (pml - nbs) after their scaffold protein promyelocytic leukaemia protein (pml, also known as trim19 or myl). The pml gene consists of nine exons and alternative splicing yields six nuclear pml isoforms (pml i - vi) and one smaller cytoplasmatic isoform (pmlvii). However, of these seven isoforms there are several alternative spliced variants yielding at least 18 different forms of pml3 . At the conserved n - terminus, pml contains a motif that defines the trim (tripartite motif) family of proteins . It is characterized by an rbcc motif composed of a conserved ring domain, one or two b - boxes, and an -helical coiled - coil domain 4, 5 . The ring domain is a cysteine rich domain with zinc bound in a cross - brace conformation . This structure is ideal for the formation of larger protein aggregates and therefore crucial in processes such as cell growth, oncogenesis, apoptosis, and rna trafficking during viral infections 5, 6 . The b - box provides the correct orientation and alignment of the coiled - coil domain 7 . When correctly positioned, the coiled - coil domain is responsible for homo and hetero - dimer interactions between trim proteins3 . The rbcc motif of the pml protein makes it possible to sustain a highly dynamic turnover of partner proteins during the cell cycle 8, 9 which could explain the functionally promiscuous nature of the pml - nbs . Pml is not only responsible for maintaining the integrity of the complex but also for the recruitment and correct localization of all pml - nb proteins, e.g. P53, sp100, daxx and cbp 4 . Sumo-3 attachment has been reported to be responsible for the correct nuclear localization of pml 10, and without sumo1 modification several protein partners are not recruited to the pml - nb 10 - 12 . However, unsumoylatable mutants of pml (3kr) still show the typical nuclear speckle pattern 11 indicating that also other factors play a role in pml - nb formation . Recently, pml was identified as the first protein degraded by sumo - dependent polyubiquitination after treatment with arsenic 12 . Sumoylation is a post - translational modification where one of the four isoforms of the small ubiquitin - like modifier (sumo) protein is conjugated to a target protein as a single sumo molecule (sumo1) or in polymeric chains (sumo2/3)13 . The enzymatic cascade is similar to the ubiquitin pathway14 (figure 1). However, unlike ubiquitination, which targets proteins for degradation, sumoylation modifies proteins to interact in nuclear transport10, 15, transcriptional regulation14, 16, apoptosis17, 18 and protein trafficking19 . Sumo is synthesized as a precursor and its conjugation requires exposure of two c - terminal glycine residues . The processing of immature sumo is one of the three functions of sumo specific proteases (senps). In addition, senps are also capable of dismantling sumo chains and reversing sumoylation by cleaving the isopeptide bonds between sumo and its protein targets or between the several sumos20 . Thus far, there are six known senp isoforms (senp1 - 3 and senp5 - 7)20 with different assignments in the cascade (table 1). After maturation, sumo is activated in an atp dependent manner by thio - ester bond formation with the e1 activation proteins uba2/aos114 . The activating e1 enzyme uba2p was first identified in yeast where it is, together with aos1, necessary and sufficient for smt3p (yeast homologue of sumo) activation in vitro 30 . Subsequently sumo is transferred from uba2 to the sumo conjugating enzyme ubc9 14, 32 . Ubc9 recognizes the consensus sequence kxe where is a large hydrophobic amino acid and k is the lysine for sumo conjugation33 . Ubc9 is believed to be the only sumo conjugating enzyme32, 34 and sumoylation of its binding partners is enhanced by interaction with p14arf35 . Finally, an isopropyl bond between the terminal glycine of sumo and the -aminogroup of lysine of the target protein is formed by an e3 ligase . Several unrelated proteins have been attributed e3 sumo ligase activity: nucleoporin protein ranbp236, topors37, 38, several pias proteins39 - 41, polycomb group protein pc242, and rnf443 . Recently an anti - sumo ubiquitin - protein isopeptide ligase (e3) has been described: mel-18 interacts both with hsf2 and the sumo e2 ubc9 and inhibits ubc9's activity which decreases hsf2 sumoylation44 . Because of the many associated proteins, pml - nbs are involved in a wide variety of cellular processes such as dna damage and repair45, 46, apoptosis47 - 49, senescence50, 51, cell cycle52, transcription53, carcinogenesis54 and virus infection55, 56 . Despite the involvement in such vital processes, pml deficient mice are normal, fertile and do not have a higher occurrence of tumors 57 . Although the actual function of the bodies still has to be determined, three non - mutually exclusive hypotheses about the function of pml - nbs have been proposed in literature . The first hypothesis gives pml - nbs a role as a nuclear depot thereby providing a mechanism for regulating nucleoplasmic levels of proteins in which pml bodies release proteins as they are needed58 . Since nascent rna has been found in the proximity of the pml - nbs59 and pml - nbs seem to associate in regions with high transcriptional activity60, 61, the second hypothesis proposes that pml - nbs are associated with nuclear activity such as gene transcription and dna replication 62 . Indeed, most well - described partners of pml - nbs such as p53, daxx, mdm2 and sp100, all have transcription activation or repression activity 4, 49, 63 - 66 . Furthermore, pml - nb structure is dependent on the integrity of the surrounding dna . In fact, many factors which accumulate in pml - nbs are known histone methyltransferases, histone deacetylases or dna methyltransferases, suggesting that pml - nbs may play an important role as epigenetic regulator of transcription and replication (for a review see 67 and references therein). The third hypothesis is that pml - nbs provide a nuclear platform for post - translational modification (ptm) such as phosphorylation 68, 69, acetylation70 and sumoylation71 . These three hypotheses support the concept of dynamic interactions with pml and a high turnover of protein associates, instead of a static complex with a fixed number of partners . The involvement of pml - nbs in such a variety of processes has led to an enormous research interest in this enigmatic protein which has culminated in an overwhelming number of publications . In recent years, several techniques such as affinity capture, yeast two hybrid, co - localization and mass spectrometry have been employed to identify proteins of the pml complex which could lead to a better understanding of the pml - nb function . This reductionist approach has successfully identified several of the components of the pml - nbs but added more pieces to the puzzle instead of narrowing the spectrum of possible functions . Systems biology approach could be essential to grasp the true complexity of the pml - nbs and to allow us to generate hypotheses based upon the vast amount of information available in distributed sources . Not all pml partners described in literature are well represented in protein databases such as intact72, molecular interactions database (mint)73, biogrid74, human protein reference database (hprd)75 and nuclear protein database (npd)76 . This paper attempts to bridge the gap between (the lack of) database content and literature information . An integrated view of all the existing information is indispensable to gain true understanding of the functionality of large protein complexes such as pml - nbs . We present a curated network model of pml - nbs based on information extracted from protein interaction databases (mint, intact, biogrid, hprd and npd) and fragmented information obtained through extensive literature search . For the compilation of the network we used the open source network analysis software cytoscape . It features high flexibility and the possibility to analyze protein networks with various (downloadable) plug - in software extensions allowing us to analyze and interpret the collected pml network . Based on this model we offer a system - wide perspective on the pathways and proteins that gather at the nuclear bodies . The network is publicly available (proteomics division at http://www.ua.ac.be/ppse) to make it a central information resource for pml - omics researchers . A manually curated pml - nb interaction network was established in cytoscape77 by integration of pml interaction data retrieved from databases and literature (figure 2). First we assembled a primary pml - nb network established with data retrieved from protein databases i.e. Mint73, biogrid74, hprd75, npd76 and intact72 . Second, we expanded the primary network by adding pml - nb components which were exclusively found through extensive literature (only peer - reviewed journals) search . Finally, to create a biologically relevant network, interactions between pml - nb associated proteins were retrieved with the aid of cytoscape import tools . Via the uniprotid we searched the intact and cpath database 78 to further complete the network . After importing all available data from these databases the information was confirmed or completed by searching the biogrid, hprd and mint databases also using the uniprotid . Interactions found by cpath and intact exploration which could not be verified in other databases were manually checked in literature . To compile the network, pml was the only protein used as pml - nb determinant . Often sp100 or daxx are used as pml - nb determinants but these proteins also dynamically move in and out of the pml - nbs59 . Interesting is the presence of a sub - network displaying alt - associated - pml nuclear bodies (apbs) (figure 2, boxed). These special pml bodies are only seen in a subset of interfase nuclei in telomerase - negative immortalized human cells . These cells maintain their telomeres by a mechanism known as alternative lengthening of telomeres (alt). Thus far, apbs have not been observed in mortal cells or telomerase positive immortal cells 79, 80 . In the apb, pml co - localizes with telomere binding proteins trf1 and trf2, replication protein a, rad50, rad51, rad52, rap1, mre11, tin2 and nbs1/p9579, 81 - 84 . For rad51 and rad52 there is no evidence yet but all other components, including pml are required for apb formation 85 . It is suggested that pml is required for the transcriptional activation of p53 in alt cells and thus induces apoptosis via the p53 pathway86 . After integrating the pml - nb components from database search and literature the network contains 166 nodes (proteins) and 781 edges (interactions). To provide an overview of the characteristics of the pml - nb components and interactions which connect these we provide several edge and node attributes (table 2). The criteria used to annotate these attributes are standardized and the order in which several sources are used is strictly maintained . Whereas in most publications only the most common pml - nb partners are mentioned, this is the first time pml - nb components are brought together in a comprehensive network . Depending on the researcher's interest, the network can be analyzed from different angles, eventually using additional tools such as cytoscape plugins . We analyzed the general properties of the pml - nb components using the attributes described above in table 2 . The network contains 166 protein associates of pml of which 70 are described in literature but are not available in any of the standard protein interaction databases . The majority (57%) of the pml - nb proteins are nuclear, 33% of the pml - nb interactome shuttles actively between nucleus and cytoplasm, while 9% of the components are predominantly cytoplasmatic . To provide an overview of the processes in which pml - nbs are involved, we categorized the uniprot function keywords in groups and distributed the 166 pml - nb partners in these groups . As expected, most of these proteins appear to be nuclear or nucleus / cytoplasm shuttles . Another interesting and well represented group involves proteins which are part of the virus host interaction . In addition, pml nuclear bodies have been implicated in processes as apoptosis and cell cycle it is not surprising that many of the protein partners are directly involved in these processes . An interesting group is the one of the small protein conjugation, e.g. Ubiquitin and sumo, which is found under the information embedded in the edges illustrates the diversity of techniques underlying the integrated data that have been used to detect interactions with pml . Affinity capture, in vitro reconstitution of protein complexes and co - localization by immunofluorescence are the most commonly used methods to detect interactors of the pml - nbs . Further research will demonstrate if components found with these methods interact binary with pml . Less frequently used are the two - hybrid methodology or biochemical assays, two techniques which do in fact prove binary interactions with pml . Of the total number of pml - nb components (166), 59% of the interactions one must therefore keep in mind that false positives may be present in the network . Experiments were performed in diverse cell types which range from commonly used immortal lines such as hela, mcf-7 and hek293 to primary cell cultures . Using several cell lines gives an overall idea of the composition of the pml - nb which is relevant for integration in systems biology and contributes to the knowledge of pml - nb composition in different cell types . The possibility exists that we adopt certain proteins as part of the complex while this architecture is unique for a certain cell type (e.g. Alt cells). Equally important to create a uniform view of the pml protein complex is the necessity to clearly state -if possible- which since pml has 6 different splice variants with different kinetics 9, it is expected that these isoforms may (partially) bind other partner proteins . In only one third of the cases therefore, to determine isoform - dependent differences in pml - nbs it is vital that researchers clearly state which pml variant is used in their experiments whenever possible . Apart from the direct interactions with pml, we imported the links between the several pml partners from the intact database to show distinct biological pathways that meet at the pml - nbs . Hence, next to the direct edges (166) with pml, these 166 partners are interconnected by 615 edges (figure 2). Upon analyses of the functions of the pml - nb components, a significant number of mostly nuclear proteins seems to be involved in ubiquitin - like modification processes such as sumoylation and ubiquitination (figure 3, ptm (small protein)). Due to the close relationship between pml sumoylation and nb formation10, 11, 94, 95 and because of pml - nbs may act as a platform for post - translational modifications; we explored the possibility if the pml - nbs could be sumoylation hotspot in the nucleus . Pml - nbs are a hub for numerous proteins and are involved in a range of functions . Moreover, the complex is highly dynamic and proteins such as cbp and sp100 show a dynamic exchange between nuclear bodies and nucleoplasm96 . We wondered if the dynamics of the complex would be related to sumoylation of nb proteins . Thus, the pml - nbs would be a (de)sumoylation platform where sumo is coupled or detached to / from its target protein . Possible evidence confirming this hypothesis lies in the observation that a large number of confirmed sumoylated proteins localizes at the nbs . In fact, apart from sumo4, all sumo isoforms are found in the pml - nbs . Furthermore, not only several senps were co - localized with pml but also activating enzyme uba297, the only sumo conjugating enzyme ubc998, 99 and several sumo ligases such as ranbp2 and piasy 100 - 103 (figure 4). One of these, ranbp2, promotes pml sumoylation 104 . In addition, another pml - nb component, hdac7, has been described to exert sumo e3 ligase - like activity and also enhances sumo modification of pml 105 . In fact, the most interesting sumo e3 ligase at the pml - nbs could be pml itself . Indeed, in yeast, the ring domain of pml is sufficient for pml auto - sumoylation and sumoylation of other proteins 106 . Moreover, in transfected mammalian cells, pml ring mutants show a dramatical decrease in pml auto - sumoylation 107 . In vitro, when purified pml is incubated with hela lysate, ubc9 and sumo1, this leads to auto - sumoylation 98 . However, one must keep in mind that, although all these observations suggest a sumo e3 ligase activity for pml, indirect effects (e.g. Stimulation or increased expression of enzymes from the sumoylation machinery) may also be involved and further research is required to define pml as a novel sumo e3 ligase . Dynamics between sumoylation and de - sumoylation seems important for the architecture and function of the nuclear bodies . It has been shown that il6 cytokine induces the upregulation of mrna expression of senp1 . Augmented senp1 expression causes pml de - sumoylation which abolishes the suppressor activity of pml on stat3 109 . On the other hand, suppression of senp1 senp2 hydrolyses sumo1 from pml in vitro and changes the cellular localization of pml protein partners such as cbp, daxx and p53111 . Further on, truncated senp5 co - localizes in the pml - nbs instead of in the nucleolus and both truncated- and wild type - senp5 show the ability to remove sumo2 or sumo3 from pml lys160 or lys49026 . Finally, senp6 suppression results in the accumulation of sumo2/3-modified species in pml bodies 29 but these observations suggest that not only pml is influenced by the presence of senps but that they also have an effect on other pml - nb targeted proteins . All the necessary components to attach sumo to a target are present at the pml - nbs but also the senps to remove sumo again . This means that the complete cycle of sumoylation and de - sumoylation could take place at the pml - nbs (figure 4). In fact, pml contains a sumo binding motif which forms a non - covalent interaction with sumo attached to protein partners107 . Possibly these proteins are recruited to make a functional switch at the pml - nbs by detachment of sumo . A large amount of pml partners have been described in literature as sumoylated (38%). However, this percentage may well be an underestimation because sumoylation of many pml partners has not been investigated yet . Therefore, to have an idea about other possible sumoylation targets in the network we searched for pml - nb proteins with an negatively charged amino acid - dependent sumoylation motif (ndsm) motif as predicted by yang et al.91 . In fact, more than 56% of the pml - nb components contain an ndsm motif of which about half was already found to be sumoylated in wet lab experiments . Complementary to existing knowledge of sumoylation sites, we employed an in - house developed machine learning approach to predict genome - wide sumoylation sites . We used the method based on conditional random fields that previously proved successfully to predict kinase specific phosphorylation sites112 . A 9 residue sequence window surrounding the annotated sumoylation sites from uniprot was used to train a conditional random field - based model as described in dang et al . (2008)112, with a feature function template optimized with a genetic algorithm as described in dang et al . Known sumoylation sites were extracted from uniprot (downloaded august 18, 2009) based on the sequence feature " crosslink " and a feature description containing the string " sumo " . Feature annotations containing the " probable " or " by similarity " qualifiers were removed . The performance of the training step was assessed by k - fold (k=4) cross validations . In four fold cross validation, the protein set is partitioned into four subsets, of these subsets a single subset is retained as validation data for testing the model . This process is repeated four times with each of the four subsets used exactly once as the test set . Its excellent performance is demonstrated by an area under the curve (auc) value of 0,91 . Also in figure 5 is a venn - diagram of the overlap of what is described in literature and what is predicted . In general there is a large overlap between our screening method and the search for ndsm motifs91 . We determine fewer proteins as sumoylation target compared to the ndsm paper, which is determined by the stringency of our threshold . Figure 5b demonstrates that the overlap between literature and our approach and the overlap between the ndsm predictions and literature is equal, even though yang et al . Found more unconfirmed targets . Interestingly, there appears to be a portion of targets which were not predicted by our method or the method of yang et al ., suggesting that there are other motifs and surrounding amino acids which characterize a sumoylation target . To asses a potential enrichment of sumoylated proteins at the pml - nbs, percentages of predicted sumoylated proteins were compared between the full human proteome and the pml - nb . To accomplish this, the trained model was used to predict potential sumoylation sites in the full human proteome (using the uniprot sequences), with a predefined maximum allowed false positive rate (fpr) of 1% as described in dang et al.112 . Figure 5c gives an overview of the predicted percentages of sumoylatable proteins in several cellular compartments . Genome wide about 24% of the protein sequences contained at least one predicted potential sumoylation site . Within proteins annotated with the go cellular component terms " cytoplasm " and " nucleus ", respectively 26% and 33% were predicted to contain one or more sumoylation sites . Within the assembled pml network, 48% contained 1 or more predicted sumoylation motifs, demonstrating a clear overrepresentation of sumoylation motifs in the pml network that cannot solely be attributed to its nuclear localization . Since our predictor is trained with all possible known sumoylation sites, its predictions are not focussing on one particular motif (such as the ndsm motif) but on all possible motifs . Together with the excellent performance demonstrated by the roc curve and the stringent thresholds applied, we believe that this is the most reliable approach to be able to draw conclusions from the intracellular distribution of predicted sumoylation sites . The observed discrepancies between our predictor and the ndsm results may, besides algorithmic performance differences, also reflect the diversity of the training sequences and the stringency of the performed prediction . Dynamic complexes such as the pml - nbs require high - throughput methods as well as specialized reductionist approaches for characterization and identification of all the participating components . As much, the pml community should be cautious for scattered data making it difficult to maintain a general overview . We believe that our pml network helps in giving a comprehensive insight in the vast amount of pml data that has been generated . Creating this overview focused the attention on a role of the pml - nbs in sumoylation . The presence of the enzymes for both sumoylation and de - sumoylation suggests that both the attachment and detachment of sumo can take place at the pml - nbs . In fact, the properties of the pml protein such as the ring domain and coiled coil motif are ideal to recruit unmodified proteins which are targets for sumoylation . Amongst others, pml itself may act as an e3 sumo ligase . In addition, pml contains a sumo binding motif that can bind sumoylated proteins which might be in turn de - sumoylated . To estimate if there was an enrichment of possibly sumoylated proteins at the pml - nbs, independent of cellular localization, we used an in - house prediction method for a genome wide screen for sumoylatable proteins . Although in the nucleus this number rises slightly to 33% we found that 48% of the pml - nb components contain one or more possible sumoylation sites . Taken together the presence of (de)-sumoylation enzymes combined with an enrichment in sumoylatable proteins suggests that pml - nbs are a hotspot for nuclear sumoylation where protein targets meet the components of the (de)-sumoylation pathway . The dynamic regulation of key point proteins at the pml - nbs supports this hypothesis since the regulatory function of sumoylation on pml - nb related processes such as transcription 14, 113, 114, cell cycle115, apoptosis116, senescence117 and viral infection 118 - 120 is slowly uncovered . As an example we present in table 3 the influence of sumoylation on pml - nb related transcription factors . Further, this hypothesis could be the glue between the three previously formulated hypotheses about pml - nb function . Since sumoylation is often related to a functional switch, it is not unthinkable that by sumoylation certain protein factors are retained in the nucleus until they are needed and are released by de - sumoylation or vice versa . This sumoylation dependent switching could meticulously regulate specific nuclear processes such as transcription and dna related processes . In fact, our functional analysis reveals a very high number of transcription factors confirming the close relationship between pml - nbs and the modulation of transcription, possibly in a sumo - dependent manner . Similarly, several protein partners are involved in dna replication, damage sensing and repair which indicate a role in dna related processes including epigenetic regulation 67 . The pml - nbs can thus be seen as a nuclear relay station where protein fractions involved in a variety of nuclear processes make a sumoylation regulated functional switch . Increasingly, the crosstalk between sumoylation and other ptms such as acteylation and phosphorylation (e.g. Via the pdsm sumoylation motif 92) is uncovered 121, 122 . Since at the pml - nbs also kinases, acetyltransferases and methyltransferases gather, it is likely that we are just exploring the borders of the regulatory complexity within this protein complex.
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A 19-year - old patient was admitted for low back pain and radiating pain in the posterior aspect of the left leg which had begun 2 months prior . She was treated with nonsteroidal anti - inflammatory drugs, physical therapy, and an epidural block in the local hospital . She mainly complained of pain in the left leg, with a score of 6/10 on the visual analogue scale (vas), and a result of 50 degrees in a passive straight leg - raising test . There were no sensory - motor abnormalities, and the tests for reflexes showed normal responses . Magnetic resonance imaging (mri) revealed central herniation of the l4-l5 intervertebral disc, which compressed the dural sac (fig . Discectomy was suggested, as the patient complained of consistent and severe pain, the mri findings showed severe spinal stenosis, and there were no responses to conservative treatment . Therefore, we decided to treat her with percutaneous epidural neuroplasty with a racz catheter on the left l5 spinal nerve root . After obtaining her informed consent, prophylactic antibiotics (ceftezole 1 g) were administered intravenously after negative skin test confirmation . Routine monitoring equipment - including an electrocardiogram, blood pressure monitor, and pulse oximeter - were applied, and she was placed in the prone position on the fluoroscopy table . A pillow was inserted under the abdomen to correct the anteflexion of the lumbar vertebrae, the legs were abducted, and the feet were inverted . Following sterile preparation and draping, the location of the sacral hiatus was confirmed by fluoroscopy, and a local infiltration of 1% lidocaine was performed in the insertion area (1 inch right lateral and 2 inches caudal from the sacral hiatus). The tip of the needle was placed between the s3 and s4 vertebrae, whose locations were confirmed by fluoroscopy with a c - arm . After confirming negative aspiration of the blood or cerebrospinal fluid (csf), 10 ml of water - soluble contrast media were injected for the epidurogram . The racz catheter was inserted through the needle and was advanced towards the left l5 nerve root under continuous fluoroscopy (fig . 2). 1,500 units of hyaluronidase in 10 ml of normal saline were then injected . Additional dye (2 ml) was injected to confirm the contours of the nerve root, and 9 ml of 0.2% ropivacaine and 1 ml of 4 mg dexamethasone were injected following negative aspiration . The patient did not complain of severe pain, paresthesia, or unusual responses during the procedure . After 1 hour, the patient complained of sudden motor weakness in the right lower limb, on the site opposite to where the neuroplasty was conducted . The great toe dorsiflexion was marked as motor grade i, and the ankle dorsiflexion as motor grade ii . Paresthesia and a dull sensation in the right l4-l5 dermatome had developed, but the patient did not complain of pain . At first, we suspected a temporary motor weakness caused by ropivacaine, and the symptoms were observed for one hour . An urgent mri was performed, but no epidural hematoma or suspicious lesions were found (fig . Around 4 hours after the neuroplasty, the motor and sensory weakness had not improved; emergency surgery was therefore performed . Discectomy of the l4-l5 intervertebral disc was conducted . During the surgery, central and left paracentral protrusions of the disc were found, and swelling of the right l5 nerve root was also discovered . After the surgery the motor strength and sensory function of the right leg had returned to normal . In the 10-day follow - up period, there were no signs of motor or sensory abnormalities, and the patient was therefore discharged without any discomfort . Bleeding, infections, or nerve damage can generally develop after epidural neuroplasty, but motor and sensory weaknesses are rare complications . Acute monoplegia following epidural adhesiolysis has previously been reported in a patient with failed - back syndrome and foraminal stenosis of the l4-l5 levels . However, to the best of our knowledge, there has been no report of acute motor weakness in the opposite lower extremity after percutaneous epidural neuroplasty . First, motor weakness can be caused by direct nerve injury from the epidural needle or racz catheter during the procedure . However, this was unlikely in this case, as there were no complaints of acute severe pain, sensory abnormality, or abnormal responses during the procedure . This was also unlikely in this case, as these injections cause bilateral or patchy blocks of the motor and sensory functions . Fourth, although nerve damage from hypertonics can be a reason, we did not use hypertonic saline . Finally, injection of a large volume of fluid into the epidural space may cause a transient neurological deficit . Rocco et al . Reported that epidural injections after spinal surgery may cause complications such as cauda equine syndrome or nerve root damage as the epidural space after surgery is limited, and the injected drugs may increase the pressure and cause nerve damage either directly or by ischemia, due to decreased blood flow . They presented acute right monoplegia which had occurred after epidural adhesiolysis of the bilateral l5 nerve root in a patient with failed - back surgery syndrome, with spontaneous recovery after 5 weeks . They suggested that the large volume of fluid injection might have been the reason for the transient neurological deficit, as a compartment loculated by the fluids might have compressed the nerve . They also suggested careful injection under observation of the excretion of the contrast dye through the neural foramen in order to prevent nerve injuries caused by compression from a large volume of injected drugs . In particular, if the spinal stenosis is severe, the epidural injection may increase the pressure in the epidural space, and even a mild edema around the nerve root may cause a neurologic deficit by compression of the nerve root . We hypothesized that the large volume of fluid injected during the epidural neuroplasty and the severe spinal stenosis might have been the reason for the opposite motor weakness in this case, as per the previous reported case . The patient in the present study presented severe spinal stenosis, with a herniated disc occupying more than 2/3 of the spinal canal . The epidurogram during the neuroplasty showed a better spread of the contrast dye in the left side, and a filling defect on the right side . Moreover, the central protrusion may have increased the pressure on the right side of the epidural space, and the closed compartment may have caused loculation, leading to barotrauma in the right l5 nerve root . In addition, swelling of the l5 nerve root was observed during the surgery, and the motor and sensory functions returned to normal immediately after the surgery, suggesting that the latter had a decompressive effect . However, further evaluations, including measurement of the pressure and radiologic tests, are required to prove this theory . In conclusion, we presented a rare case of unilateral motor weakness following epidural neuroplasty, on the side opposite to the lesions . The injection of a large amount of fluid during neuroplasty in a patient with spinal stenosis may cause compressive nerve damage.
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Everyday intentionally or unintentionally we plan the treatment options for our patients care . To draw these clinical decisions, we almost rely on the wealth of our own experience in clinical practice, discussion with health professionals or academicians, literature from the text books, journals and recent or current review articles . As academicians it is very important to assist our best credibilities to teach the students and also to make substantial clinical decisions to carry out the research projects . Oral health professionals need to focus on the quality of the research journals and keep updated about the current critical reviews . Thus by this way of learning, any health professional can be able to good from poor research provided he himself is updated about the literature and methods . These are the types of studies, where the data is collected from the subjects either in the form of a questionnaire, personal interviews, telephonic interviews or via online services like web or e - mail networks . The results of the survey done with the help of the questionnaires are always subjective and generally depends on the response gained from the respondents . The most valuable assert for conducting a medical research are the health professionals . Gaining the research experience from the health professionals is much different than the experience gained by the general population . Globally, several studies assessed the value of qualitative data collection for research in dentistry . In addition, this health professional survey aimed to analyze the response of al - farabi colleges academic staff's interest and experience in research, their knowledge of different study designs and its application to find out the objectives behind any research study . The questionnaire was simplified and constructed based on the classification suggested by the international epidemiological design criteria . Bachelor / master / doctorate (phd) minimum 2 years of experience in university . Fresh graduateshealth professionals working in hospitals and public health sectorshealth professionals working part time in academics . Health professionals working in hospitals and public health sectors health professionals working part time in academics . The questionnaire tool [table 1] was developed using a published questionnaire from a previous study . The authors (sar, ok) revisited the questions to adopt the study objectives . Academicians working at al - farabi colleges were randomly selected to participate in the pilot study . Several changes were carried out to some questions to improve its clarity and to reflect the discrepancies in the nature of bds, msc and phd holders . The al - farabi colleges institutional research review committee requested few modifications to the questionnaire and provided ethical approval for the study . Research tool (questionnaire) the validated questionnaire was comprised of two pages . It was divided into three parts each of which consisted of five questions in addition to demographic section [table 1]. The three variables are (1) research experience, (2) study design, (3) objectives and common designs . Each question three hundred questionnaires were sent to the teaching faculty members recruited at al - farabi colleges male / female campuses as bds, msc and ph.d . Via personal contact and by electronic mail service . The e - mail consists of a 15-item questionnaire with an informed consent and brief outline towards the research protocol . The respondents were asked to fill the questionnaire with their consent and provide their inputs by ticking the options yes or no for the first two variables, tick the option for the last variable . Two reminders were sent by e - mail to all the nonresponders 4 week subsequently after the initial mailing, if not responded then they were termed as nonrespondents for our study . The statistical analysis was performed using spss package version 20, ibm . The cross tabulation to examine the association of the gender and qualification was done using the chi - square tests . The knowledge scores of the respondents by qualification and gender were obtained using anova and t - tests . Bachelor / master / doctorate (phd)minimum 2 years of experience in university . Bachelor / master / doctorate (phd) minimum 2 years of experience in university . Fresh graduateshealth professionals working in hospitals and public health sectorshealth professionals working part time in academics . Health professionals working in hospitals and public health sectors health professionals working part time in academics . The questionnaire tool [table 1] was developed using a published questionnaire from a previous study . The authors (sar, ok) revisited the questions to adopt the study objectives . Academicians working at al - farabi colleges were randomly selected to participate in the pilot study . Several changes were carried out to some questions to improve its clarity and to reflect the discrepancies in the nature of bds, msc and phd holders . The al - farabi colleges institutional research review committee requested few modifications to the questionnaire and provided ethical approval for the study . Research tool (questionnaire) the validated questionnaire was comprised of two pages . It was divided into three parts each of which consisted of five questions in addition to demographic section [table 1]. The three variables are (1) research experience, (2) study design, (3) objectives and common designs . Each question three hundred questionnaires were sent to the teaching faculty members recruited at al - farabi colleges male / female campuses as bds, msc and ph.d . Via personal contact and by electronic mail service . The e - mail consists of a 15-item questionnaire with an informed consent and brief outline towards the research protocol . The respondents were asked to fill the questionnaire with their consent and provide their inputs by ticking the options yes or no for the first two variables, tick the option for the last variable . Two reminders were sent by e - mail to all the nonresponders 4 week subsequently after the initial mailing, if not responded then they were termed as nonrespondents for our study . The statistical analysis was performed using spss package version 20, ibm . The cross tabulation to examine the association of the gender and qualification was done using the chi - square tests . The knowledge scores of the respondents by qualification and gender were obtained using anova and t - tests . A total of 95 completed questionnaires were included in the analysis amounting the final response rate to 31.66% . Furthermore, the respondents were 16 people from the bds group, 61 from the msc group and 18 from the ph.d . Group . About the first variable - research experience, descriptive frequencies showed a similar response when all the three groups were compared . The bds group gave a positive response (yes) value of 93.50%, msc had a value of 91.80% and phds were at 88.90 . According to the data analysis in table 2a, the qualification showed that the positive response ranged from 76 to 100% . Was much higher for the bds group (100%) as compared to the msc (90.2%) and ph.d . The entire msc and ph.d . Groups (100%) claimed to have conducted clinical and experimental studies (q4) compared to their bds (68.8%) counterparts . When both the genders were compared, females claimed to have a much higher experience (100%) in conducting clinical studies as compared to males (90.9) and it showed a statistical significance of (0.071). Group thought that the research conducted these days could be manipulative, and this was statistically significant (p <0.005). Objectives and common designs second variable (study design and research criteria): descriptive frequencies were found to be low for the bds (38.90%) and msc (66.23%) groups, but the ph.d . Group showed a relatively higher frequency (72.22%). According to the data analysis in table 2b, the respondents showed a lack of knowledge, with the correct answers ranging from 57% to 67% . Only two - third of the respondents were aware of the study design, there was no significant statistical difference between the academic degrees . A significantly higher percentage of ph.d . Respondents (77.8%) were aware that the case - control studies were associated with the presence or absence of a disease (q4) as compared to the msc group (62.3%) and the bds group (18.8%). When the data were analyzed according to the gender there was no statistical difference seen . Third variable (objectives and common designs): descriptive frequencies were lower for the bds group (23.75%), but the msc and ph.d . Likewise [table 2c], all the respondents showed poor knowledge of the objectives and the common designs, the difference being highly significant for all the questions when the data were analyzed according to the academic qualifications (p <0.005). When the answers were analyzed according to the different genders, nearly a statistical significance (0.061) was seen with respect to the first question . Table 3 shows a significant association of knowledge with gender and qualification (bds, msc and phd). The total mean of knowledge (10.77 1.70) with significant association with gender and academic degree . Knowledge scores of the respondents by qualification and gender the bds group showed the least knowledge scores when compared to the msc and ph.d . Male respondents showed better knowledge scores (11.20) when compared to the females (10.20) with a significance value (p <0.005). Our study intended to gauge the opinion and response of the health professionals associated with academics to their research experience, design, and the objection and commonly used designs in the field of research . The data obtained from the respondents was less than satisfactory, as out of 300 questionnaires sent to the academicians via personal contacts and mail, only 95 questionnaires were complete and the participation was found to be only 31.66% in our study, we found that males responded earlier as compared to the females . According to our first variable table 2a, the response was found to be similar among all the health professionals irrespective of whether they belonged to the bds / msc or / ph.d . The second variable [table 2b] was the response rate, wherein the bds group gave a very poor response, and the msc group showed a better response when compared to the ph.d . Groups . When the data for the third variable [table 2c] was collated, the bds group showed a poor response once again, but the response of the msc and the ph.d . We found that our study was similar to the studies done by adeyemi et al . And mcsherry et al . Surveyed the opinion of resident doctors in nigeria on the use of qualitative research design and methods in dentistry . All 20 dental residents were familiar with qualitative research design, 50% residents categorized qualitative research design as social science, 25% residents said it was just a science and 25% residents were not sure of the qualitative research technique . Adeyemi et al . Admitted that the qualitative approach to research in dentistry may be useful to understand meanings which patients attach to actions, decisions, beliefs and values concerning their care . Mcsherry et al . Conducted a descriptive quantitative study about research awareness in evidence based practice . Out of his convenience sample of 2126 registered health care practitioners (rhcps), 843 rhcps responded to the questionnaires . Seven hundred and thirty - three (91%) rhcps agreed that evidence - based practice played a large role in improving patient care . This point was reinforced when 701 (86%) of respondents strongly agreed that evidence - based practice was the way forward to change clinical practice . The author concluded that irrespective of position or grade, the rhcps had a positive attitude towards research but faced many obstacles . The key obstacles listed were a lack of time, support, knowledge and confidence . During our research, we received 35 partially filled questionnaires from health professionals thus showing a lack of interest to respond . The knowledge scores of the bds group when compared to the msc and the phd groups showed a high statistical significance . The knowledge scores for the msc group compared to the phd group showed no statistical significance [table 3]. The msc graduates are well aware of the recent advances in research methodologies . In a few countries, a mini thesis is included in the msc 2 year program while in a few others a complete dissertation is included in the master of dental surgery (mds) 3-year program which is equivalent to a phd thesis . In this study, we expected the third variable to be more pronounced among the phd graduates, but it was similar to the msc graduates . The third variable showed statistical significance when the data were analyzed on the basis of qualification [table 2c]. This was evident for the qualification as the third variable had difference among the opinion for the questions . The same was evident in our study as we found a high statistical significance with bds group compared to the msc and the ph.d . Groups . The health professionals must involve themselves into evidence - based learning and supported each other by their research experience and knowledge, then a coordinated approach would enable them to gain confidence in academics or clinical practice . In our study, we found men (18.3%) responded earlier than women (13.3%). This was in agreement with the study done by mcfarlane et al ., where among 1592 of his respondents with physician demographic characteristics, 80% were male, and 20% were females . The knowledge scores [table 3] for the males as compared to the females was slightly higher and was statistically significant . This might be due to the lesser no of female academicians in the university as compared to men . The probable cause may be due to the academicians associated with the universities having varied work schedules and being preoccupied with pending questionnaire studies to answer . A few professionals like the phds are hard to reach and are redirected to their receptionists or assistants ., where he addressed the receptionists as gatekeepers because most of the questionnaires would be retained by them and hence would remain unanswered . Cummings et al . Revealed in 2001 that the response rate among the health professionals would be 10% lower than the studies conducted with the general population . In our study, we too observed a lack of response from the health professionals . According to leece et al ., 2004, he concluded, that in surveys conducted via internet or mail service, a greater nonresponse bias may be seen in comparison with other methods like personal contact via questionnaire, interviews or repeated telephonic surveys . According to mcmohan 2003, he concluded that the response accuracy in his study was low, as he found a few incomplete answers compared to the answers in the same questionnaires sent by fax or postal services . This was in conjunction with our study, as we also obtained a less than satisfactory response, as we also chose the web services via the internet e - mail service to distribute our questionnaire . (2000), suggested that the nonresponse bias can be overcome by advertising the authors with prepaid incentives, personalized letters and professional organization sponsorship could show an increased response rate as compared to sending repeated questionnaire via mail services . In this study, irrespective of the sample size, we were keen to collect the data from these learned individuals and to gauge their opinion . The questionnaire was very simple and contained very straight forward questions, thus reducing the bias with the questionnaire . Thus, we could label our research as a feasibility study, as we conducted our study by using very simple and flexible methodology . On the other hand, a pilot study uses a more rigid methodology such as sample estimation, randomization, case and control group selection and statistical analysis . Moreover, almost all pilot studies would report their results inaccurate and may demand to conduct further larger studies . The objective of our survey was to ascertain the opinion of health professionals and also their response and enthusiasm regarding this study . Our study seemed to be a qualitative research, as it showed us the actual response rather than what was expected . Thus, this kind of research is totally dependent on the participants to investigate any scientific or social cause . Most authors can interpret multiple opinions with qualitative research, but most of their experiences regarding qualitative research are strong and encouraging . Leedy and ormrod (2001) stated that qualitative research is less structured in the description, as it helps in the formation and builds up new theories via subjective responses from the participants . Unlike qualitative research, quantitative research builds on deductive reasoning . Although started in ancient times around 1250 ad, quantitative research was used for surveys and experimental studies with existing theories . Quantitative research is always independent of the researcher, as it creates the results through objectivity uncovered in the collected data . We believe that this study is limited due to the low response rate, and it has been conducted at the single academic institute . We envisage this study could be utilized for a broader research study that would include several academic institutes in saudi arabia and gulf region to reflect the views of academic staff on this important notion . This survey was done to judge or elicit the response of health professionals associated with academics . The analyzed data presented a high statistical significance when the bds group was compared with the mds and ph.d . It is always believed that knowledge is the most powerful tool and knowledge from research must be applied to clinical practice, thus helping us to guide the students in a better way . The art of literature criticism is a skill that can be learned, and the practice of this art will contribute to the knowledge of the literature.
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Fossil fuels have been the primary energy source of mankind since industrialization, while in the last decades sustainable and environmentally benign alternative energy sources attract increasing attention in research . Solar light can become one of the main sources of sustainable energy in the future . However, the intermittent nature of solar energy requires scalable technologies for its harvesting and converting into storable and transportable energy carriers . In this regard, production of liquid or gaseous chemical fuels (e.g., h2, formic acid, alcohols, or light hydrocarbons) by solar - energy - driven water splitting and carbon dioxide reduction is seen as a promising option . Production of these so - called solar fuels can be facilitated by a suitable photocatalytic material that combines light harvesting and redox functions . Typical photocatalysts are inorganic semiconductors, and titanium dioxide is the most widely studied material . This wide band gap (3.03.2 ev) oxide finds application in water splitting, dye - sensitized solar cells, and photocatalytic remediation of pollutants . The energy conversion efficiency of most semiconductor - based systems remains low (in most cases <1%) due to a mismatch between the lifetime of photogenerated charge carriers (femtoseconds to nanoseconds) and slow kinetics of the redox reaction (milliseconds to minutes), resulting in severe electron hole recombination . Therefore, understanding of the semiconductor bulk and interfacial charge carrier dynamics and how they couple to surface redox processes is key to optimizing photocatalytic systems . Over the last decades, titania has become a guinea pig of photocatalytic research, and the mixed - phase commercial pigment aeroxide p25 (ca . 7580% anatase, 2520% rutile) is an example of an extensively studied tio2-based material that is frequently used as benchmark photocatalyst . The charge carrier dynamics in tio2-based materials have been investigated by different spectroscopic techniques such as transient microwave conductivity (trmc), photoluminescence (pl), transient absorption spectroscopy (tas), electronic paramagnetic resonance (epr), and stimulated desorption . These methods provide information about charge carrier trapping and recombination (trmc, tas, stimulated desorption) as well as the nature (epr) and depth (pl) of the trap states . Among them, infrared spectroscopy has proven to be a versatile tool since it can simultaneously assess light - induced changes of surface groups and adsorbates and the majority charge carriers . Over the last decades, steady - state and time - resolved ir spectroscopy was used to study electrons in n - type tio2 . Both free conduction band and shallow trapped electrons (cbe and ste, respectively) interact with electromagnetic irradiation and give rise to specific electronic absorption in the ir region . Absorption (a) due to cbe is featureless, and its intensity exponentially increases with the decrease of the probing light energy:1where is the frequency of the probing light in wavenumbers, k is the proportionality coefficient, and n is an exponent which can take values between 1.5 and 3.5 depending on the electron scattering mode . Optical transitions due to ste can be described, for instance, as excitation of electrons from shallow donor states into the conduction band continuum:2where e is the energy of ir irradiation in electronvolts, k is the proportionality coefficient, eop is the energy of the ir light required to excite an electron from the shallow state into the conduction band continuum, and ethermal is the thermal ionization energy of the donor state . Even though charge carrier dynamics in tio2-based materials have been extensively studied over the last decades, there are still some discrepancies regarding the role of surface hydroxyls and adsorbed water in trapping and recombination of photogenerated charge carriers . For instance, it has been reported that dehydroxylation of tio2 at elevated temperatures slows down the decay of photogenerated electron absorption from minutes to hours . On the contrary, a recent time - resolved ir study revealed that water adsorbed on nanocrystalline anatase increases the yield of photogenerated electrons and slows down their recombination at the picosecond to nanosecond time scale due to trapping of photogenerated holes by adsorbed water . In keeping with this, showed that surface - trapped holes in tio2 only exist in the presence of adsorbed water . In this work we investigated how hydration of tio2 p25 affects the dynamics of photogenerated electrons at the millisecond to minute time scale in order to better understand its effect on charge carrier trapping and recombination . To this end, we used rapid scan time - resolved diffuse - reflectance infrared fourier transform spectroscopy (drifts). We found that dehydration of tio2 at t> 423 k accelerates charge carrier recombination and leads to a reversible hysteresis of charge carrier dynamics . Oxidative treatment altered the hydration state of tio2 p25 and induced formation of a prominent ste signal and slowed down its decay rates . Temperature - dependent measurements revealed low activation energies of the electron absorption decay rates in hydrated samples . Ab initio dft+u analysis of the holes trapped on anatase tio2(101) revealed that they are more stable and mobile on hydrated surface than in the dehydrated system . These findings demonstrate that adsorbed associated water stabilizes surface - trapped photogenerated holes in tio2 p25 and suppresses charge carrier recombination at the millisecond to minute time scale characteristic for chemical reactions . Commercial mixed - phase tio2 (aeroxide p25, evonik industries) was used in all experiments . The morphology of the material was characterized by bright field transmission electron microscopy with an fei technai g2 (type sphera) electron microscope operated at 200 kv . X - ray diffractograms were collected on a bruker endeavor d2 powder diffractometer equipped with a bragg brentano goniometer, a cu cathode, and a 1d lynxeye detector (nickel - filtered k, 1.0 mm primary beam slit and 3.0 mm beam knife height). Diffuse - reflectance uv vis spectra were recorded with a shimadzu uv-2401pc uv vis spectrometer equipped with an integrating sphere accessory, using baso4 as reference . A bruker vertex 70v ftir spectrometer equipped with a praying mantis diffuse - reflectance accessory (harrick scientific) and a liquid - nitrogen cooled mct detector was used for the diffuse - reflectance infrared fourier transform spectroscopy (drifts) measurements . The ir measurements and sample treatment were carried out in an in situ drifts low - temperature reaction chamber (harrick scientific) equipped with two kbr and one fused - silica windows for ir signal collection and sample irradiation, respectively . The cell was connected to a home - built gas delivery system . During all experiments, the temperature of the cell exterior was maintained at 293 k by cooling water . The sample compartment of the spectrometer was purged with dry nitrogen during all experiments . The spectra were recorded at 4 cm resolution in the spectral range of 3950600 cm . Steady - state spectra reported in this work are averages of 100 scans . A low - pass ir filter (cutoff frequency 3950 cm) was placed in front of the detector compartment to block stray light and reflected laser irradiation . Kbr (sigma - aldrich, ir grade) was used as reference for the survey drift spectra . Typically, the spectrum of the sample in the dark was used as the reference for difference spectra and in time - resolved measurements . The following settings were used: 4 cm spectral resolution, 40 khz scanner velocity, acquisition of single - sided interferograms at forward and backward mirror movement with automatic splitting of the interferograms by bruker opus 7.5 software . With these settings, temporal profiles of the ir signal at particular wavenumbers were extracted by bruker opus 7.5 from the 3d data blocks consisting of transient spectra stacked against the delay time . The signal - to - noise ratio was poor, results of several independent measurements were averaged during data processing . O2 (99.95%), h2 (99.999%), and ar (99.999%) gases were supplied by linde and passed through moisture and/or oxygen filters (agilent technologies). Exposure of the sample (60 s for time - resolved measurements) was controlled by a software script and an optical shutter (thorlabs). More details on data conversion and processing can be found in the supporting information (cf . Also figure s1). Untreated tio2 (sample i) was prepared by keeping as - delivered tio2 p25 in the spectroscopic cell under dynamic vacuum (p <10 mbar) for 45 h at 293 k prior to the experiments . Room - temperature dehydrated tio2 (sample ii) was prepared by keeping as - delivered tio2 p25 at 293 k under dynamic vacuum for 36 h. dehydrated titania (sample iii) was prepared by heating tio2 to 623 k for 12 h under dynamic vacuum followed by cooling to room temperature under static vacuum . Rehydrated sample (sample iv) was prepared with the same procedure as sample iii but the sample was kept for 16 h in 300 mbar dry ar after cooling down to 293 k. residual water in the ar stream (<0.1 ppm) and water desorbed from the cell interior rehydrated the sample during this hold time . The oxidized sample (sample v) was prepared by heating titania to 523 k under dynamic vacuum, followed by irradiation with a 325 nm laser (10 mw / cm) under static vacuum for 1 h. subsequently, the reaction chamber was filled with 50 mbar o2 and the sample was irradiated with the uv light again until a stable spectrum was obtained . After this, the reaction chamber was evacuated, refilled with 50 mbar o2, cooled to room temperature, and then the sample was exposed to uv light for 30 min in the presence of oxygen . The resulting material (sample v) was stored in 300 mbar o2 overnight in dark prior to further spectroscopic experiments . The color of tio2 samples did not turn blue or gray upon prolonged evacuation and/or heating, which indicated that no substantial reduction of the studied materials took place under the chosen experimental conditions . All calculations were performed using the vienna ab initio simulation package (vasp). The ion electron interactions were represented by the projector - augmented wave (paw) method and the electron exchange - correlation by the generalized gradient approximation (gga) with the perdew burke sham valence states were expanded in a plane - wave basis set with a cutoff energy of 400 ev . We have used the dft+u approach, in which u is a hubbard - like term describing the on - site coulombic interactions . This approach improves the description of localized states in titania, where standard lda and gga functionals fail . The values of u(ti) = 4.2 ev and u(o) = 5.25 ev were applied to the ti 3d and o 2p states, which have been used previously to successfully localize the electrons and holes in n- and p - type defect states in titania, in agreement with experimental epr data . Our calculated gga + u lattice parameters for anatase titania were a = 3.907 and c = 9.724 . The most abundant and most stable facet exposed in tio2 p25was modeled by a periodic slab using a 3 3 supercell consisting of seven layers of in total 63 tio2 units . The bottom three layers were frozen to their bulk position, and the four top ti o twelve water molecules were adsorbed on anatase tio2(101) surface to simulate the hydrated interface (figure s2, supporting information). Due to the large cell size the climbing image nudged - elastic band (ci - neb) algorithm was used to identify the transition states for the trapped hole migration on the anatase tio2(101) surface . The dark survey spectrum of untreated p25 tio2 (sample i) is shown in figure 1 . 3400 cm (broad) and 1622 cm correspond to the stretching and bending vibrations of adsorbed associated water, respectively (cf . The presence of hydroxyls on the anatase and rutile phases was evident from the bands at 3716, 3693, 3680, 3658, and 3631 cm . The bands in the 29802840 cm region indicate the presence of hydrocarbon moieties, while those at 1554 and 1439 cm can be attributed to oxygenates and/or carbonates . These organic compounds resulted from adsorption of ambient contaminants on as - delivered tio2 p25 . Left: room - temperature dark drift spectra of tio2 p25 in static vacuum: untreated sample i (black), dehydrated sample iii (red), oxidized sample v (blue). H stretching region . Note: the spectra are vertically offset for convenience . When the untreated sample i was exposed to 325 nm irradiation, a broad featureless absorption band developed in the 29001000 cm region while the ir bands of adsorbed water and surface hydroxyls decreased in intensity (figure 2). Intensity of the featureless absorption band increased toward lower wavenumbers and could be reliably fit with function 1 characteristic for cbe . The best fit was achieved with n = 1.6 0.1 in good agreement with previous reports . This band was hardly affected by addition of ar, while molecular oxygen substantially quenched its intensity (figure s3, supporting information), which further confirms its assignment to photogenerated electron . (sample i) in a static vacuum under 325 nm irradiation (black curve) and the exponential fit of the experimental data (red curve). H stretching region . Similar to previous reports, the decay rates of the photogenerated electron absorption band were wavenumber - independent in the 29001000 cm range . Therefore, we monitored its transient behavior at 1205 cm, where no molecular bands were present (figure 3, black curve). The electron absorption decay in the dark showed a complex behavior . Within the first minute the signal intensity decreased to ca . 30% of its initial value and then decayed to zero at much slower rates . A reliable fit of the initial part of the kinetic curve was achieved with either a second - order decay or a sum of two exponents . Both models gave similar r values and half - life times of the signal decay: (11.8 0.3) s (table 2). 1 min onward followed first - order kinetics with a half - life time of 2 min . No reliable fit of the kinetic curves in the broad time range (5 min) could be achieved when only a second - order or a two - exponential model was used, but rather a sum of first- and second - order decays or a three - exponential model was needed tio2 p25 samples subjected to different treatments: black, untreated sample i; red, sample iii dehydrated at 623 k; blue, rehydrated sample iv; green, oxidized sample v. inset: normalized decay curves of untreated and rehydrated samples . Temporal profiles were recorded after 60 s of exposure to 325 nm irradiation at 293 k under static vacuum . For details on sample treatment such a complex decay of the photogenerated electron absorption may stem from different phenomena . First, if the observed signal originates from well - defined single species (i.e., cbe), then faster and slower decay components can correspond to at least two different charge carrier recombination channels . For instance, a faster second - order decay can correspond to bimolecular recombination of trapped electrons and holes, while a slower first - order component may be due to defect - assisted recombination . Second, both cbe and ste may contribute to the broad absorption band formed in the low - wavenumber region under uv irradiation (figure 2). In this case, faster and slower decay components may arise from the recombination of photogenerated holes with cbe and ste, respectively . Finally, a combination of both situations cannot be excluded which severely complicates the development of a reliable analytical model . In this study we opted for the sum of first- and second - order decays to fit the data over a broad time range, while the second - order decay kinetics was used to fit fast initial decay of the signal (for more details on the fitting procedure see supporting information). Have previously reported that dehydroxylation of tio2 p25 slows down the decay of photogenerated electrons from minutes to hours . On the other hand, a recent study of photogenerated electron dynamics at the picosecond to nanosecond time scale shows that adsorbed water suppresses charge carrier recombination in anatase tio2 . In order to evaluate whether adsorbed water has a positive or an adverse effect on the charge carrier recombination at the millisecond to minute time scale, we studied photogenerated electron absorption dynamics in the material which was dehydrated under dynamic vacuum (p <10 mbar) at 623 k (sample iii). The room - temperature dark drift spectrum of this sample is shown in figure 1 (red curve). As expected, for this sample the intensities of adsorbed water and surface hydroxyls bands were substantially lower than for untreated tio2 . H stretching region of sample iii contained two well - resolved bands at 3716 and 3673 cm, while the bands at 3680, 3658, and 3631 cm were almost absent (figure 1, red curve). The bending vibration band of adsorbed water at 1620 cm, which was absent in the spectrum recorded at 623 k (figure s4, supporting information), reappeared upon cooling, but with lower intensity than before the treatment . The broad band of hydrogen - bonded hydroxyls and associated water (3400 cm) was diminished to a weaker well - resolved feature at 3460 cm . This band can be assigned to the asymmetric stretching mode of water molecules present in the form of small clusters (table 1). The difference spectrum formed in sample iii under 325 nm irradiation is shown in figure 4, black curve . Uv excitation decreased intensities of the bands at 3618, 3460, 3090, and 1621 cm, while a well - resolved band at 3690 cm and a broad feature in the 1600900 cm region emerged . The band at 3618 cm was due to surface hydroxyls of rutile tio2, while the 3460 and 1621 cm bands corresponded to stretching and bending vibrations of adsorbed water, respectively . The band at 3690 cm can be ascribed to water dissociatively adsorbed on anatase and/or rutile tio2 (table 1). The broad absorption feature formed in the region below 1600 cm could be fit with function 1, and its intensity was quenched in the presence of o2, confirming its cbe origin . Compared with sample i (figure 2), this band had lower intensity, its onset shifted toward lower wavenumbers, and it decayed at substantially faster rates . The half - life decay time decreased from (11.8 0.3) s to (2.9 0.5) s upon tio2 dehydration (table 2). Moreover, in the dehydrated sample cbe absorption decayed to zero after already 30 s in the dark without a prominent slow component (figure 3)., our results demonstrate the adverse effect of dehydration on the charge carrier lifetime in tio2 p25 . Difference room - temperature drift spectra of dehydrated sample iii (black) and rehydrated sample iv (red) tio2 p25 in static vacuum under 325 nm irradiation . When sample iii was left in the in situ drifts low - temperature reaction chamber in 300 mbar ar for 16 h, it was partially rehydrated by residual water in ar (<0.1 ppm) and water desorbed from the internal cell walls (sample iv). This was evident by the bands at 3675, 3655, 3630, and 3400 cm which reappeared in the ir spectrum but with lower intensities than before (figure s5, supporting information). Both the difference spectrum produced in sample iv by 325 nm irradiation (figure 4, red curve) and the decay rates of the photogenerated electron absorption (figure 3, inset) were similar to those of the untreated material (figure 2), except for the intensities of these spectral features . This suggests that the amount of water adsorbed on titania strongly affects the apparent electron absorption intensity (i.e., the number of photogenerated charge carriers) but not the room - temperature charge carrier dynamics . The reversible effect of dehydration and slower charge carrier recombination rates found for the hydrated samples (table 2) demonstrate that adsorbed water substantially slows down electron the above - discussed experimental results show that dehydration of tio2 adversely affects both the intensity of photogenerated electron absorption and its lifetime . On the other hand, adventitious organic adsorbates the untreated tio2 p25 exhibited vibrational bands at 29802840, 1554, and 1439 cm (figure 1) due to hydrocarbons, organic oxygenates, and/or carbonates (table 1). In order to remove these adventitious adsorbates we oxidized the material at 523 k in 100 mbar o2 . When the sample was heated in the dark, the intensities of the bands at 29802840 cm decreased, a new band emerged at 2349 cm, and the bands at 1554 and 1439 cm became stronger . These spectral changes evidenced oxidation of adsorbates containing hydrocarbon moieties to gas - phase co2 and carboxylates (table 1). After about 1 h, the intensities of these changes became stable and were not affected by the addition of oxygen or increase of the treatment time . The remaining hydrocarbons and formed carboxylates were further oxidized to co2 when the material was irradiated with 325 nm light at 523 k in 100 mbar o2 . When the resulting sample was cooled to room temperature either under static vacuum or in dry ar, its behavior under uv irradiation did not differ from that of the non - oxidized dehydrated material . This suggests that adventitious organic adsorbates had no significant effect on photogenerated electron dynamics at the millisecond to minute time scale . Unlike adventitious organic adsorbates, changes of tio2 hydration had a very prominent effect on the photogenerated electron absorption intensity, spectral appearance, and decay rates . When the oxidized sample was cooled to room temperature in 50 mbar o2 instead of ar or vacuum (see previous section), exposed to 325 nm irradiation for 30 min, and stored overnight under the same o2 atmosphere (sample v), the survey drift spectrum exhibited stronger bands of surface hydroxyls and adsorbed water compared with untreated titania (figure 1, blue curve). H stretching region of sample v showed a prominent band at 3693 cm of water dissociatively adsorbed on tio2 . This band was also present in untreated titania but only as a shoulder (figure 1). Other differences between samples i and v included a decreased intensity of the 3631 cm band and appearance of the band at 3670 cm . The latter band was rather broad and could include superimposed 3680 and 3658 cm bands . These changes agreed well with the notion that water adsorbs more easily on anatase tio2 under uv irradiation in the presence of o2 than in an inert atmosphere . When sample v was exposed to 325 nm irradiation the bands of adsorbed water (3400, 1631 cm) and surface hydroxyls (3693, 3681, 3670, and 3634 cm) decreased in intensity (figure 5). This was accompanied by the appearance of the band at 3734 cm which was attributed to terminal hydroxyls of the anatase tio2 and was not observed in the untreated sample (figure 2). I the bands at 3633 and 3667 cm were affected the most, while in sample v the most prominent intensity change was observed for the 3671 and 3693 cm bands (insets in figures 2 and 5, respectively). Yet the most striking difference between these two samples was the intensity and the structure of photogenerated electron absorption bands . Compared with the untreated material, the band formed in sample v had higher intensity and did not follow exponential function 1 but almost linearly increased toward lower wavenumbers evidencing formation of species other than cbe . Difference room - temperature drift spectrum of oxidized p25 tio2 (sample v) in static vacuum under 325 nm irradiation . The electron absorption formed in oxidized sample v under uv irradiation (figure 5) was similar to the bands previously observed by panayotov et al . The authors ascribed such bands to the overlapping absorption of the cbe and ste components . Here, we adopted the model developed by panayotov et al . To fit our experimental spectra (figure s5, supporting information). From this fitting, one can see that a substantial part of the signal corresponds to ste . This accounted for the stronger photogenerated electron absorption signal observed in sample v and may explain its slower decay rates in comparison with untreated titania (table 2). Despite the substantially different decay rates, the absorption signal decay profiles of sample v exhibited a complex behavior similar to that of the untreated and rehydrated samples (figure 3). First, the signal decayed to ca . 25% of its initial value following the apparent second - order decay kinetics with 0.5 = (62 2) s. then, the remaining absorption relaxed to zero in an apparent first - order process with 0.5 = (3.6 0.5) min (table 2). Previous sections show that both the intensity of photogenerated electron absorption and its decay rates depend on tio2 hydration . However, room - temperature measurements alone are insufficient to derive a reliable conclusion about the role of adsorbed water in the photogenerated charge carrier dynamics . This is because both cbe and ste can contribute to the apparent electron absorption (figure s5, supporting information). Moreover, the studied samples exhibited a number of stretching o h bands whose positions and intensities were obscured by hydrogen bonding (table 1). In order to understand how adsorbed water affects photogenerated charge carrier dynamics we studied the electron absorption decay rates as a function of temperature and pretreatment (figure 6). Temperature dependence of the characteristic electron absorption decay times found for the tio2 p25 samples subjected to different treatments: (a) untreated sample i; (b) rehydrated sample iv; (c) oxidized sample v; (d) sample ii dehydrated at room temperature for 36 h; (e) sample iii dehydrated at 623 k; (f) untreated sample i at t <273 k. values in electronvolts represent apparent activation energies derived from the linear regions of the plots (red lines). Independent from the treatment, all hydrated samples showed two distinct regimes of the electron absorption decay . In the 293423 k temperature range, the apparent decay rates exhibited arrhenius - type behavior, while they did not vary with temperature above 423 k (figure 6a d). It is worthwhile to mention that, above 423 k, the bands of associated water at 3400 and 1622 cm, as well as the o h bands at 3632 and 3683 cm, substantially decreased in intensity in comparison with lower temperatures (figure s4, supporting information). The dehydrated sample iii showed no apparent temperature dependence of the charge carrier recombination rates in the temperature range of 323723 k (figure 6e). Above 523 k the drift spectra of sample iii exhibited mainly the bands of terminal hydroxyls at 3720 and 3670 cm . Below this temperature the 3460 cm band emerged in the survey drift spectra . This band was attributed to stretching vibrations of water adsorbed in the form of small clusters, and it was stable for at least 34 h after cooling to 293 k (figure s6, supporting information). Eventually, the broad absorption feature of associated water with the maximum at 3400 cm and the bands at 3693, 3670, and 3630 cm reappeared in the survey drift spectra evidencing rehydration of titania . Rehydration restored properties of the material under 325 nm irradiation both at 293 k (see section 3.2) and elevated temperatures (figure 6b). Hence, we surmise that the hysteresis of the photogenerated charge carrier dynamics observed in tio2 upon its heating above 423 k related to the association of adsorbed water . This is because sample iii exhibited fast temperature - independent charge carrier recombination (figure 6e) while its survey drift spectra evidenced the presence of terminal hydroxyls and small clusters of adsorbed water (figure s6, supporting information) but not the associated water band . The latter indicates that neither terminal hydroxyls nor small water clusters can efficiently suppress electron these findings agree with a recent epr study showing that surface trapping of photogenerated holes is inefficient in anatase tio2 dehydrated at 423 k. from figure 6, one can see that the apparent activation energies of the photogenerated electron absorption decay were almost identical for the untreated and rehydrated samples (0.080.09 ev) and slightly lower for the sample dehydrated at room temperature (0.06 ev). Although oxidized titania exhibited the slowest electron absorption decay rates and the highest apparent activation energy of 0.17 ev in the 293423 k temperature range, charge carrier recombination was temperature - independent above 423 k (figure 6c) similar to other hydrated samples . Consequently, differences in apparent activation energies and the electron absorption decay rates can be attributed to different hydration of the materials rather than their intrinsic properties . From the temperature - dependent plots (figure 6a d) one can expect that cooling of the hydrated samples below 293 k can further slow charge carrier recombination . Contrary to this, we found that the photogenerated electron absorption decay rates were temperature - independent between 263 and 153 k, yet slightly faster than at 293 k (figure 6f). The intensity of electron absorption at these temperatures was substantially lower than at 293 k. besides this, steady - state spectra of untreated tio2 changed when it was cooled below 273 k. the absorption bands at 3400 and 1620 cm became weaker, the shoulder at 3695 cm disappeared, and the 3720 cm band grew in intensity compared with the sample at 293 k (figure s8, supporting information). As water could not desorb from titania under these conditions, the aforementioned spectral changes can be explained by perturbed interaction between the oxide surface and adsorbed water . For instance, the increase of the terminal hydroxyls band intensity at 3720 cm can be interpreted as weakened hydrogen bonding between adsorbed water and surface hydroxyls . Even though the apparent activation energies obtained in this work are difficult to assign to a specific process, these values, when combined with the effect of dehydration and low temperature, provide valuable phenomenological insights about the interplay of surface hydration and charge carrier dynamics in tio2 p25 at the millisecond to minute time scale . First of all, these apparent activation energies are unlikely to directly correspond to water desorption . This is because the desorption energy is at least 0.6 ev and increases with decreasing water coverage (i.e., hydration) which does not agree with our findings . The hysteresis of the charge carrier dynamics observed upon dehydration / rehydration of titania and rather low apparent activation energies suggest that association of the oxide with adsorbed water can account for these phenomena . When temperature increases, hydrogen bonding among adsorbed water molecules and between water and oxide becomes weaker . This destabilizes trapped charge carriers, increases their recombination rates, and results in the apparent activation energy . The absence of apparent activation energy for the electron absorption decay at temperatures below 273 k (figure 6f) can be understood by assessing the strength of hydrogen bonding through the shifts of the stretching o h frequencies . As the o h bands positions and intensities of sample i were almost identical at 153 and 263 k (figure s8, supporting information), hydrogen bonding is expected to be similar in both cases . When association does not change with temperature the electron our experimental results showed that the decay rates of photogenerated electron absorption in tio2 p25 depend strongly on the hydration state of titania and hydrogen bonding between the oxide and adsorbed water . These findings suggest that the slowed down charge carrier recombination rates in hydrated n - type tio2 originate from the interaction of adsorbed water with surface - trapped holes rather than with electrons . This is because all tio2 samples exhibited hysteresis of the electron absorption dynamics at temperatures above 423 k irrespective of the nature of photogenerated electrons (figure 6). For instance, the photogenerated electron absorption signal in the oxidized sample v was dominated by ste up to 523 k. nonetheless, the electron absorption decay rates in this sample were temperature - independent above 423 k. this hysteresis can be understood by taking into account the recent finding of panarelli et al ., who observed that the surface - trapped holes disappear from the epr spectra when water was desorbed from tio2 at 423 k. this was interpreted in terms of stabilization of surface - trapped holes by adsorbed water present on the oxide . Suppressed charge carrier recombination at the picosecond time scale due to trapping of photogenerated holes by water adsorbed on anatase nanoparticles these observations agree with the expected interaction of adsorbed water molecules with photogenerated holes that are driven to the surface in hydrated tio2 . In order to better understand how associated adsorbed water interacts with photogenerated holes in the studied titania samples and to this end, we studied the migration of a photogenerated hole trapped on the anatase tio2(101) surface by an ab initio dft+u analysis . 84 wt% of used tio2 p25 and (101) is the most stable and abundant anatase facet . The dehydrated sample was represented by a clean tio2(101) surface while the hydrated material was modeled by adding 12 associated water molecules to the system . Some of these water molecules adsorbed dissociatively forming surface hydroxyls . The (101) surface exhibits three types of oxygen atoms: twofold - coordinated bridge oxygen (o2c), threefold - coordinated surface (o3c(a)), and threefold - coordinated subsurface (o3c(b)) atoms, as well as sixfold (ti6c)- and fivefold (ti5c)-coordinated titanium (figure s2, supporting information). The surface - trapped hole (h) the formed charge was compensated with a uniform background of charge of the opposite sign, and then the system was allowed to relax . In the dehydrated system the hole localized on o2c had 0.28 ev lower energy than on a o3c(b) site, while no stable configuration was found for the hole trapped on the o3c(a) atom . For the hydrated system the situation was substantially different . Namely, the most energetically favorable localizations of the hole were o2c (figure 7, parts a and d) and o3c(b) (figure 7, parts c and e) atoms (the latter was only 0.03 ev lower in energy), while the hole localized on o3c(a) was ca . 0.5 ev higher in energy than either of these two sites . When the hole was localized on the o3c(b) atom, the corresponding ti5c o bond broke, which resulted in the formation of a fourfold - coordinated ti atom . In the dehydrated system this atom was thermodynamically unstable . On the other hand, water adsorbed on the ti5c site stabilized this undercoordinated ti atom in the hydrated system . The bond rearrangement upon migration of h from the o2c site to o3c(b) site in the hydrated system is shown in figure 7, parts d and e. the ti5c o3c(b) bond length was 2.08 and 2.31 when the hole localized on the o2c and o3c(b) sites, respectively . These differences in hole localization on clean and hydrated anatase tio2(101) agreed well with the works of selloni and co - workers . Migration of the hole (h) trapped at the anatase tio2(101) surface along the transport coordinate as defined in figure s2b . (a and c) spin density of h localized into the o2c and o3c(b) sites . (b) spin density of the transition state for h migration from o2c to o3c(b) site . (d and e) configurations of h localized into the o2c and o3c(b) sites . (f) potential energy surfaces of h migration on the tio2(101) surface with and without adsorbed water . In order to evaluate whether adsorbed water affects mobility of the hole trapped on the anatase tio2(101) surface we chose this trajectory because the alternative path would involve the o3c(a) atom which was energetically substantially less favorable for the h localization on both clean and hydrated surfaces . First of all, we found that the transition state was shared between o2c and o3c(b) atoms (figure 7b), which agreed well with a previous study of the hole transfer from anatase tio2(101) to a surface hydroxyl . The barriers for the hole migration from o2c to o3c(b) in dehydrated and hydrated systems were 0.31 and 0.17 ev, respectively . Beside the higher barrier, hole migration on the clean tio2(101) surface involved the unfavorable o3c(b) site (figure 7f). From this, one can expect a substantially higher mobility of the surface - trapped holes in hydrated titania compared with dehydrated samples . This higher mobility can also account for lower charge carrier recombination rates observed for hydrated tio2 . Besides this, the difference between the theoretical barriers of the hole migration in dehydrated and hydrated systems was ca . 0.10.2 ev (figure 7f), in keeping with the experimental apparent activation energies (figure 6). These results support our hypothesis that the apparent activation energies obtained in the present work for the hydrated samples originate from the hydrogen - bond interaction between the adsorbed water and titania . Moreover, the difference between the surface - trapped hole mobility in dehydrated and hydrated systems can account for the hysteresis of the charge carrier dynamics upon dehydration of tio2 (figure 6). The influence of tio2 p25 hydration on the decay rates of photogenerated electron absorption at the millisecond to minute time scale was investigated by rapid scan time - resolved drift spectroscopy . Dehydrated titania exhibited a weaker electron absorption signal which decayed at higher rates compared with hydrated samples . The slowest electron absorption decay was found for tio2 after an oxidation treatment . The photogenerated electron absorption feature formed under 325 nm uv laser irradiation in untreated and oxidized tio2 was dominated by contributions from conduction band electrons (cbe) and shallow trapped electrons (ste), respectively . Charge carrier recombination rates in hydrated samples increased with temperature in the 293423 k range with low apparent activation energies between 0.06 and 0.17 ev . The electron absorption decay rates became temperature - independent at t> 423 k and t <273 k. at high temperature a perturbed interaction between adsorbed water and the oxide surface resulted in the temperature - independent charge carrier recombination, while below 273 k this was due to constant strength of hydrogen bonding between titania and adsorbed water . The experimental results were complemented by an ab initio dft+u analysis of surface - trapped hole migration on dehydrated and hydrated anatase tio2(101). This analysis revealed that adsorption of water stabilizes holes localized on surface oxygen atoms and lowers hole migration barriers . These effects are expected to increase charge carrier mobility and with that suppress electron we conclude that the apparent activation energies obtained in this study originate from hydrogen - bonding interactions between adsorbed water and tio2, which in turn are involved in the stabilization of surface - trapped holes leading to the observed prolonged lifetime of photogenerated electrons.
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Bulking agent injection is now a standard treatment for patients with grade 1 - 3 primary vesicoureteral reflux (vur). Patients with higher grades and secondary types of vur show an unacceptably high failure rate when treated with injections of a bulking agent . Collagen has been used for many years as a bulking agent in ureterovesical orifice and bladder neck to treat vur and incontinence . Collagen, however, can get calcified in the tissue, a process that can cause a series of problems for patients . We present a case of a patient in whom calcified injected collagen showed imaging features mimicking ureteral stone . A 15-year - old girl presented with moderate degree of right flank pain for 2 weeks . Pain was localized on the right side, altered with change in position, and did not relate to any particular organ . The patient had been treated successfully for bilateral grade 2 vur when she was 3 years old . A kidney ureter bladder (kub) radiography was requested and the report indicated presence of bilateral ureteral stone [figure 1]. Ultrasonography showed bilateral ureterovesical junction (uvj) stone and mild stasis in both kidneys, which was compatible with the findings of the non - enhanced spiral abdomino - pelvic computed tomography (ct) scan [figure 2]. However, diethylene triamine pentaacaetic acid (dtpa) scan with furosemide injection and radionuclide cystography (rnc) scan ruled out obstruction and relapse of vur . Calcification of bilateral intravesical collagen injected to treat her vur 12 years earlier was responsible for the positive findings on kub, ultrasonography, and spiral ct . 15-year - old female with bilateral flank pain, which was suspected to be due to stones in bilateral ureterovesical junction, later diagnosed as due to calcification of collagen used 12 years earlier to treat vesicoureteral reflux . 15-year - old female with bilateral flank pain, which was suspected to be due to stones in bilateral ureterovesical junction, later diagnosed as due to calcification of collagen used 12 years earlier to treat vesicoureteral reflux . Spiral ct scan of pelvis without intravenous and oral contrast shows bilateral uvj calcifications (arrows). Endoscopic therapy for vur has gained popularity because of elimination of invasive surgical procedures in children . In addition, it has an acceptable success rate and a low level of complications . Although a transition has occurred in the material of choice from teflon to collagen and now to hyaluronic acid copolymers . However, we do find patients who were treated with collagen now in the second decade of their life . The injected material, being a foreign body, can induce inflammatory response that leads to tissue calcification . This calcification has been reported to be symptomatic with some patients reporting episodes of stone passing, hematuria, and even obstruction . This is the second case report of ureterovesical injection calcification presenting as a ureteral stone during differential diagnosis and the first report of its imaging . Unlike the first case report where the patient presented with renal colic and passing of stone, our patient was completely asymptomatic, except for positional non - related flank pain . There is no difference between a ureteral stone and calcification of injected collagen, when viewed by different imaging modalities like kub [figure 1], ct scanning [figure 2], and also ultrasound examination . This similarity is because of the place where the bulking agent is injected . In doubtful cases, as in our case, uvj obstruction can be ruled out by dtpa nuclear scan in which glomerular filtration of injected nuclear radioisotope reveals any obstruction in the upper urinary tract . The diagnosis of uvj stone in the presence of uvj calcification, possibility of ureteral stone passing (in a patient with renal colic) after uvj injection therapy, and the probability of recurrent vur after ureteral intervention (in which calcified ureteral orifice will tolerate dilatation to pass the ureteroscope) remain to be studied . Vur can be assessed by rnc in which a radioisotope passing to the bladder via the urethral catheter reveals presence of vur . The standard treatment for grade 1 and 3 primary vur is injection of a bulking agent into the uvj . Clinicians should be aware that calcification of bulking agent like collagen can occur and they need to differentiate this from ureterovesical stones on imaging findings.
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Deep vein thrombosis (dvt) and pulmonary embolism (pe) are important pathologies that affect apparently healthy individuals as well as medical or surgical patients . Therapeutic objectives are essentially the prevention of thrombus extension and embolization, and the prevention of recurrent episodes of venous thromboembolism (vte) to reduce the risk of fatal pulmonary emboli . Despite the availability of different treatment strategies, the large majority of patients commonly receive a similar therapeutic approach, and the choice of the treatment is eventually influenced by the severity of the presentation of the disease . Anticoagulation is the main therapy for acute vte and the evidence for the need for anticoagulation in these patients is based on the results of clinical studies performed more than 40 years ago . Patients need to start treatment as soon as the diagnosis is confirmed by objective testing, and because anticoagulant drugs with a rapid onset of action are needed in this phase, three parenteral therapeutic options are currently available for initial treatment: unfractionated heparin (ufh), low - molecular - weight heparin (lmwh), and fondaparinux . Fondaparinux is a synthetic pentasaccharide that inhibits factor xa indirectly by binding to antithrombin with high affinity and was recommended for the first time in the 8 edition of the american college of chest physicians (accp) guidelines on antithrombotic and thrombolytic therapy, which is the most recent and was published in 2008 . This recommendation was based on the results of the matisse studies [3, 4]. In the matisse dvt study, 2205 patients with dvt were treated with a once daily subcutaneous dose of fondaparinux (7.5 mg for patients weighting 50 to 100 kg, 5.0 mg for patients weighting less than 50 kg and 10.0 mg for patients weighting more than 100 kg) or with a twice daily subcutaneous dose of enoxaparin (1 mg / kg) for at least five days . There were no differences in the incidence of recurrent vte at 3 months (3.9% vs 4.1%), major bleeding while on treatment (1.1% vs 1.2%), and mortality at 3 months (3.8% vs 3.0%). In the matisse pe study, 2213 patients with acute pe were randomly allocated to treatment with subcutaneous fondaparinux or intravenous uhf . Recurrence of vte at 3 months (3.8% vs 5.0%) and major bleeding while on treatment (1.3% vs 1.1%) were again similar between the two groups . In selected cases, there is widespread agreement that patients with pe resulting in cardiogenic shock initially treated with thrombolysis plus anticoagulation have better short- and long - term clinical outcomes than those who receive anticoagulation alone . More recently, some authors have proposed that thrombolysis should be administered to patients with normal blood pressure (and no contraindications) when clinical or echocardiographic evidence of right ventricular dysfunction is present . In the most recent accp guidelines, the use of thrombolytic therapy, which was previously recommended for hemodynamically unstable patients (massive pe) only, is now also suggested for selected high - risk patients without hemodynamic instability and with a low risk of bleeding, with a grade 2b recommendation . However, this remains a controversial issue, and the controversy is likely to remain at least until the results of an ongoing european trial, in which 1,000 pe patients with preserved systolic blood pressure, elevated troponin levels, and right ventricular enlargement on echocardiography are randomised to thrombolytic therapy (tenecteplase plus heparin) versus heparin alone, will become available . Other guidelines, such as those of the european society of cardiology, currently do not recommend routine use of thrombolysis in non - high - risk patients . As soon as possible after the diagnosis of vte, most patients are also started on oral anticoagulant treatment with vitamin k antagonists for the long - term secondary prevention of the disease . Because of their slow onset of action, and because of their potential to paradoxically increase the prothrombotic state of the patient by also inhibiting endogenous anticoagulants such as protein c, vitamin k antagonists cannot be used as the only treatment strategy during the acute phase of disease and thus require initial association with parenteral anticoagulants for a minimum of 5 days . After this period, oral anticoagulant therapy alone is continued until its benefits (reduction of recurrent vte) no longer clearly outweigh its risks (increase in bleeding). The risk of recurrence after stopping therapy is largely determined by two factors: whether the acute episode of vte has been effectively treated; and the patient intrinsic risk of having a new episode of vte . Therefore, guidelines suggest to treat vte for at least 3 months if transient risk factors are identified and to consider long - term treatment for patients with unprovoked proximal vte and no risk factors for bleeding, in whom good quality anticoagulant monitoring is achievable . When the risk to benefit ratio remains uncertain, patient preference to continue or to stop treatment should also be taken into account . Vte is defined unprovoked if cancer or a reversible provoking risk factor is not present . Reversible provoking factors include major risk factors such as surgery, hospitalization, or plaster cast immobilization, if within 1 month; and minor risk factors such as surgery, hospitalization, or plaster cast immobilization, if they have occurred 1 to 3 months before the diagnosis of vte, and estrogen therapy, pregnancy, or prolonged travel (i.e. More than 8 hours). The greater is the impact of the provoking reversible risk factor (e.g., recent major surgery) on the risk of vte, the lower is the expected risk of recurrence after stopping anticoagulant therapy . Of interest, in the most recent version of the accp guidelines, the presence of thrombophilia is no longer considered for the risk stratification of the patients . For the secondary prevention of vte in patients with active cancer, the use of lmwh for the first 3 to 6 months is now preferred over the use of vitamin k antagonists . This recommendation is based on the results of three studies that selectively enrolled a total of 1,029 patients with vte in association with active cancer and that found that, compared to oral anticoagulant therapy with vitamin k antagonists, 3 months or 6 months of therapeutic - dose lmwh was associated with less recurrent vte in one study and less bleeding in another study (relative risk for the three studies: recurrent vte, 0.56; 95% ci, 0.38 - 0.82; major bleeding, 1.01; 95% ci, 0.62 - 1.64; mortality, 0.92; 95% ci, 0.78 - 1.10) [7 - 9]. Lmwh is usually administered at full therapeutic dose for the first month and then reduced at approximately 75% of the initial dose thereafter . There is a trend toward a more extended duration (potentially life - long) of secondary prevention for a large proportion of patients with a first episode of vte, namely those with an unprovoked proximal dvt or pe who have a low risk of bleeding and those with a permanent risk factor such as cancer . Indeed, given the high rate of vte events still defined as unprovoked, which ranges between 26% and 47%, this recommendation has an enormous potential impact on the long - term management of patients with vte and on related costs . To overcome this problem, there is an increasing interest in the use of clinical prognostic factors to assist clinicians in individualizing the optimal duration of secondary prevention of unprovoked vte . These include the measurements of d - dimer and of residual venous obstruction at ultrasound . These strategies, although still not widely accepted, are now supported by the results of randomized clinical trials and of large cohort studies [11 - 16]. In the prolong study, patients with unprovoked vte underwent d - dimer testing 1 month after oral anticoagulant treatment discontinuation . Patients with a normal d - dimer level did not resume anticoagulation, whereas those with an abnormal d - dimer level were randomly assigned either to resume or to discontinue treatment . The rate of recurrences was 15.0% among the 120 patients who stopped anticoagulation as compared with 2.9% among the 103 patients who resumed anticoagulation, for an adjusted hazard ratio of 4.26 (95% confidence interval, 1.23 - 14.6). Vte recurred in 6.2% of patients with a normal d - dimer level . Because d - dimer levels may increase over time and a single normal d - dimer may be inadequate to predict a low risk of recurrence, the same group carried out a second study, the prolong ii study, with the aim to assess the time course of d - dimer and its relation with late recurrences in patients with normal d - dimer 1 month after anticoagulation suspension for a first episode of unprovoked vte . This study showed that when d - dimer becomes abnormal at the third month and remains abnormal afterward, the risk of recurrence is higher than in patients in whom d - dimer remains normal at the third month and afterward (adjusted hazard ratio: 7.9; 95% confidence interval: 2.1 - 30). Two randomized controlled studies have evaluated the role of residual vein thrombosis to predict the risk of recurrent vte [15, 16]. In the first study, patients with a first episode of dvt were managed according to ultrasound findings after an initial course of oral anticoagulant treatment . Patients with evidence of residual vein thrombosis were randomized to either stop or continue anticoagulants for 9 additional months, whereas patients without residual vein thrombosis treatment was stopped . Residual thrombosis was detected in 69.8% of patients; recurrent events occurred in 27.2% of those who discontinued (15.2% person - years) and 19.3% of those who continued oral anticoagulant treatment (10.1% person - years). The relative adjusted hazard ratio was 1.58 (95% confidence interval 0.85 - 2.93). Of the 30.2% patients without residual thrombosis, only 1.3% (0.63% person - years) had a recurrence . In the second study, 538 patients with a first episode of acute proximal dvt at completion of an uneventful 3-month period of anticoagulation were randomly assigned to fixed - duration anticoagulation (no further anticoagulation for secondary thrombosis and an extra 3 months for unprovoked thrombosis) or flexible - duration, ultrasonography - guided anticoagulation (no further anticoagulation in patients with recanalized veins and continued anticoagulation in all other patients for up to 9 months for secondary dvt and up to 21 months for unprovoked thrombosis). Overall, 17.2% of the patients allocated to fixed - duration anticoagulation and 11.9% of the patients allocated to flexible - duration anticoagulation developed recurrent vte (adjusted hazard ratio, 0.64; 95% confidence interval 0.39 - 0.99). For patients with unprovoked dvt, the adjusted hazard ratio was 0.61 (95% confidence interval 0.36 - 1.02) and 0.81 (95% confidence interval 0.32 - 2.06) for those with secondary dvt . The approach to the development of new anticoagulants as alternatives to heparins and vitamin k antagonists has been guided by the requirement for convenient administration with predictable pharmacokinetics, pharmacodynamics and a wide therapeutic window that would permit fixed dosing without requiring coagulation monitoring . Research has in particular focussed on targeting thrombin (factor iia) and factor xa, which are common to both the intrinsic and extrinsic coagulation pathways (fig . 1). Thrombin inhibitors act to prevent fibrin formation, as well as inhibiting thrombin - mediated activation of factors v, viii, xi and xiii, and platelets . Inhibitors of factor xa act at an earlier stage in the cascade, they can inhibit both free and prothrombinase - bound factor xa and are also able to inhibit clot - associated factor xa, thus preventing clot - associated factor xa from activating prothrombin and thereby contributing to the procoagulant activity of thrombi and therefore to the propagation of the thrombus . Dabigatran etexilate is an univalent direct thrombin inhibitor that binds exclusively to the active site of thrombin with the advantage, in comparison with heparins, to inactivate fibrin - bound thrombin . Moreover, dabigatran etexilate is a reversible direct thrombin inhibitor, which dissociates relatively quickly from thrombin, leaving a small amount of free, enzymatically active thrombin available for control of haemostasis . Dabigatran etexilate, is the prodrug of dabigatran, is rapidly absorbed from the gastro - intestinal tract and has a rapid onset of the anticoagulant activity, with plasma levels peak at 2 hours . Dabigatran produces a predictable anticoagulant effect, requires no coagulation monitoring and can be given once daily . It prolongs the activated partial thromboplastin time, but its effect is not dose - linear and it is not suitable for a precise quantification of the anticoagulant effect . At least 80% of dabigatran is excreted unchanged via the kidneys; therefore, the drug is contraindicated in patients with severe renal failure, with a creatinine clearance less than 30 ml / min . Dabigatran etexilate has been already licensed in the european union and in canada for the prevention of vte in patients undergoing hip- and knee - replacement surgery, with a recommended dose of 220 mg once daily for all patients but those with moderate renal insufficiency (creatinine clearence between 30 and 50 ml / min) and the elderly (aged 75 or more), for whom the recommended dose is 150 mg once daily . Dabigatran etexilate is currently undergoing a large phase iii program for the evaluation of its efficacy and safety in the acute treatment end in the secondary prevention of vte . The re - cover trial evaluated dabigatran for 6 month treatment of acute symptomatic vte, while the re - medy and the re - sonate trials are recruiting patients who have been successfully treated with standard doses of an approved anticoagulant for three to six months or who have completed 6 to 18 months of treatment with vitamin k antagonist for confirmed acute symptomatic vte, respectively . Patients with acute vte, dvt and/or pe, who were initially treated with parenteral anticoagulants, were randomized to receive dabigatran etexilate, administered at a dose of 150 mg twice daily, or dose adjusted warfarin (international normalized ratio of 2.0 to 3.0). The primary outcome of the study was the 6-month incidence of recurrent symptomatic, objectively confirmed vte and related deaths . Thirty of the 1,274 (2.4%) dabigatran patients, as compared with 27 of the 1,265 (2.1%) warfarin patients, had recurrent vte . The difference in risk was 0.4 percentage points (95% confidence interval, -0.8 - 1.5). The hazard ratio with dabigatran was 1.10 (95% confidence interval, 0.65 - 1.84). Major bleeding episodes occurred in 20 (1.6%) dabigatran patients and in 24 (1.9%) warfarin patients (hazard ratio with dabigatran, 0.82; 95% ci, 0.45 - 1.48), and episodes of any bleeding were observed in 205 (16.1%) dabigatran patients and in 277 (21.9%) warfarin patients (hazard ratio with dabigatran, 0.71; 95% confidence interval, 0.59 - 0.85). It is a potent and selective oral factor xa inhibitor with a particular chemical structure in its active - site binding region that plays a role in the oral absorption of the drug, with a relatively high bioavailabity (nearly 80%). Plasma levels of the drug peak after 3 to 4 hours, with a mean half - life ranging from 5 to 9 hours in young individuals, and from 11 to 13 hours in the elderly . The main route of excretion is renal, but the drug is also expelled via the faecal / biliar route . Rivaroxaban can be administered at a fixed dose in any patient and does not need laboratory monitoring . Also rivaroxaban has been licensed in the european union and in canada for the prevention of vte in patients undergoing hip- and knee - replacement surgery, with a recommended dose of 10 mg once daily . Two phase ii, dose - finding studies compared rivaroxaban administered at total daily doses ranging from 20 mg to 60 mg with standard therapy with lmwh followed by oral vitamin k antagonists [25, 26]. Based on the positive results of these studies, the following doses were selected for further investigation in the three phase iii clinical trials aimed to assess the acute phase and the long term treatment of dvt and pe (the einstein studies): 15 mg bid for 3 weeks followed by 20 mg qd in the ongoing einstein dvt and einstein pe studies, in which patients with objectively confirmed, symptomatic dvt or pe are randomized to treatment with rivaroxaban alone or with lmwh and vitamin k antagonists for a total period of 3 to 12 months, and 20 mg qd in the einstein extension study, in which patients who had completed 6 to 12 months of anticoagulant treatment with either vitamin k antagonists or rivaroxaban (if also enrolled in the acute phase studies) after an acute episode of vte were randomized to rivaroxaban or placebo for additional 6 to 12 months . The einstein extension study is already completed, and the results have been presented at the american society of hematology meeting in december 2009 . In this randomised, double blind, placebo - controlled study, the primary efficacy outcome was the recurrence of symptomatic vte and the principal safety outcome was the occurrence of major bleeding . During treatment, symptomatic recurrent vte events occurred in 7.1% patients treated with placebo and in 1.3% patients treated with rivaroxaban (hazard ratio, 0.18; 95% confindence interval, 0.09 - 0.39). After stopping the study medication, 1.0% symptomatic recurrent vte events occurred in both groups during the one month observational period of follow up . No major bleeding events were documented in the group of patients treated with placebo, 4 (0.7%) major bleeding events occurred in the rivaroxaban group (p = 0.106). Clinically relevant non - major bleeding occurred in 1.2% and in 5.4% patients randomized to placebo and rivaroxaban, respectively . Two (0.3%) patients in the placebo group and 1 (0.2%) patient in the rivaroxaban group died . Apixaban is an oral active factor xa inhibitor derived from razaxaban (an aminobenzisoxazole that binds to the active site of factor xa with high affinity), with superior pharmacological proprieties . It is a small molecule able to inhibit in a selective and reversible manner the active site of both free and prothrombinase - bound factor xa . Preclinical studies demonstrate that apixaban has an oral bioavailability of more than 50%: its plasma peak is achieved in about 3 h and its half - life is about 12 h . The drug is absorbed in the gastrointestinal tract, is metabolised in the liver by cythocrome - dependent and -independent mechanisms and it is eliminated through both the renal and the faecal routes . Apixaban has been assessed for the treatment of dvt in a dose finding study (botticelli dvt study). Patients were randomised to receive apixaban 5 mg bid, 10 mg bid, 20 mg od or lmwh vitamin k antagonists . The primary efficacy outcome, defined as the composite of symptomatic recurrent vte and asymptomatic deterioration in the thrombotic burden as assessed by repeat bilateral compression ultrasonography and perfusion lung scan, occurred in 4.7% of patients treated with apixaban and in 4.2% of lmwh / vitamin k antagonists treated patients . The principal safety outcome, defined as the composite of major and clinically relevant non - major bleeding, occurred in 7.3% of the apixaban treated patients and in 7.9% of lmwh / vitamin k antagonists treated patients . On the basis of this study, phase iii studies (amplify and amplify extension), testing apixaban at the doses of 10 mg and 5 mg twice daily, are now undergoing . Studies assessing the efficacy and safety of other factor xa inhibitors, such as edoxaban, are also underway . Dabigatran etexilate is an univalent direct thrombin inhibitor that binds exclusively to the active site of thrombin with the advantage, in comparison with heparins, to inactivate fibrin - bound thrombin . Moreover, dabigatran etexilate is a reversible direct thrombin inhibitor, which dissociates relatively quickly from thrombin, leaving a small amount of free, enzymatically active thrombin available for control of haemostasis . Dabigatran etexilate, is the prodrug of dabigatran, is rapidly absorbed from the gastro - intestinal tract and has a rapid onset of the anticoagulant activity, with plasma levels peak at 2 hours . Dabigatran produces a predictable anticoagulant effect, requires no coagulation monitoring and can be given once daily . It prolongs the activated partial thromboplastin time, but its effect is not dose - linear and it is not suitable for a precise quantification of the anticoagulant effect . At least 80% of dabigatran is excreted unchanged via the kidneys; therefore, the drug is contraindicated in patients with severe renal failure, with a creatinine clearance less than 30 ml / min . Dabigatran etexilate has been already licensed in the european union and in canada for the prevention of vte in patients undergoing hip- and knee - replacement surgery, with a recommended dose of 220 mg once daily for all patients but those with moderate renal insufficiency (creatinine clearence between 30 and 50 ml / min) and the elderly (aged 75 or more), for whom the recommended dose is 150 mg once daily . Dabigatran etexilate is currently undergoing a large phase iii program for the evaluation of its efficacy and safety in the acute treatment end in the secondary prevention of vte . The re - cover trial evaluated dabigatran for 6 month treatment of acute symptomatic vte, while the re - medy and the re - sonate trials are recruiting patients who have been successfully treated with standard doses of an approved anticoagulant for three to six months or who have completed 6 to 18 months of treatment with vitamin k antagonist for confirmed acute symptomatic vte, respectively . Patients with acute vte, dvt and/or pe, who were initially treated with parenteral anticoagulants, were randomized to receive dabigatran etexilate, administered at a dose of 150 mg twice daily, or dose adjusted warfarin (international normalized ratio of 2.0 to 3.0). The primary outcome of the study was the 6-month incidence of recurrent symptomatic, objectively confirmed vte and related deaths . Thirty of the 1,274 (2.4%) dabigatran patients, as compared with 27 of the 1,265 (2.1%) warfarin patients, had recurrent vte . The difference in risk was 0.4 percentage points (95% confidence interval, -0.8 - 1.5). The hazard ratio with dabigatran was 1.10 (95% confidence interval, 0.65 - 1.84). Major bleeding episodes occurred in 20 (1.6%) dabigatran patients and in 24 (1.9%) warfarin patients (hazard ratio with dabigatran, 0.82; 95% ci, 0.45 - 1.48), and episodes of any bleeding were observed in 205 (16.1%) dabigatran patients and in 277 (21.9%) warfarin patients (hazard ratio with dabigatran, 0.71; 95% confidence interval, 0.59 - 0.85). It is a potent and selective oral factor xa inhibitor with a particular chemical structure in its active - site binding region that plays a role in the oral absorption of the drug, with a relatively high bioavailabity (nearly 80%). Plasma levels of the drug peak after 3 to 4 hours, with a mean half - life ranging from 5 to 9 hours in young individuals, and from 11 to 13 hours in the elderly . The main route of excretion is renal, but the drug is also expelled via the faecal / biliar route . Rivaroxaban can be administered at a fixed dose in any patient and does not need laboratory monitoring . Also rivaroxaban has been licensed in the european union and in canada for the prevention of vte in patients undergoing hip- and knee - replacement surgery, with a recommended dose of 10 mg once daily . Two phase ii, dose - finding studies compared rivaroxaban administered at total daily doses ranging from 20 mg to 60 mg with standard therapy with lmwh followed by oral vitamin k antagonists [25, 26]. Based on the positive results of these studies, the following doses were selected for further investigation in the three phase iii clinical trials aimed to assess the acute phase and the long term treatment of dvt and pe (the einstein studies): 15 mg bid for 3 weeks followed by 20 mg qd in the ongoing einstein dvt and einstein pe studies, in which patients with objectively confirmed, symptomatic dvt or pe are randomized to treatment with rivaroxaban alone or with lmwh and vitamin k antagonists for a total period of 3 to 12 months, and 20 mg qd in the einstein extension study, in which patients who had completed 6 to 12 months of anticoagulant treatment with either vitamin k antagonists or rivaroxaban (if also enrolled in the acute phase studies) after an acute episode of vte were randomized to rivaroxaban or placebo for additional 6 to 12 months . The einstein extension study is already completed, and the results have been presented at the american society of hematology meeting in december 2009 . In this randomised, double blind, placebo - controlled study, the primary efficacy outcome was the recurrence of symptomatic vte and the principal safety outcome was the occurrence of major bleeding . During treatment, symptomatic recurrent vte events occurred in 7.1% patients treated with placebo and in 1.3% patients treated with rivaroxaban (hazard ratio, 0.18; 95% confindence interval, 0.09 - 0.39). After stopping the study medication, 1.0% symptomatic recurrent vte events occurred in both groups during the one month observational period of follow up . No major bleeding events were documented in the group of patients treated with placebo, 4 (0.7%) major bleeding events occurred in the rivaroxaban group (p = 0.106). None of these bleeding events were fatal or occurred in a critical site . Clinically relevant non - major bleeding occurred in 1.2% and in 5.4% patients randomized to placebo and rivaroxaban, respectively . Two (0.3%) patients in the placebo group and 1 (0.2%) patient in the rivaroxaban group died . Apixaban is an oral active factor xa inhibitor derived from razaxaban (an aminobenzisoxazole that binds to the active site of factor xa with high affinity), with superior pharmacological proprieties . It is a small molecule able to inhibit in a selective and reversible manner the active site of both free and prothrombinase - bound factor xa . Preclinical studies demonstrate that apixaban has an oral bioavailability of more than 50%: its plasma peak is achieved in about 3 h and its half - life is about 12 h . The drug is absorbed in the gastrointestinal tract, is metabolised in the liver by cythocrome - dependent and -independent mechanisms and it is eliminated through both the renal and the faecal routes . Apixaban has been assessed for the treatment of dvt in a dose finding study (botticelli dvt study). Patients were randomised to receive apixaban 5 mg bid, 10 mg bid, 20 mg od or lmwh vitamin k antagonists . The primary efficacy outcome, defined as the composite of symptomatic recurrent vte and asymptomatic deterioration in the thrombotic burden as assessed by repeat bilateral compression ultrasonography and perfusion lung scan, occurred in 4.7% of patients treated with apixaban and in 4.2% of lmwh / vitamin k antagonists treated patients . The principal safety outcome, defined as the composite of major and clinically relevant non - major bleeding, occurred in 7.3% of the apixaban treated patients and in 7.9% of lmwh / vitamin k antagonists treated patients . On the basis of this study, phase iii studies (amplify and amplify extension), testing apixaban at the doses of 10 mg and 5 mg twice daily, are now undergoing . Studies assessing the efficacy and safety of other factor xa inhibitors, such as edoxaban, are also underway . The current management of vte is largely based on the use of anticoagulant drugs, both parenteral drugs such as ufh, lmwh or fondaparinux for the treatment of the acute phase and oral drugs such as the vitamin k antagonists for the long term secondary prevention . All these drugs have been proven to be highly effective in preventing thrombus propagation, embolization, and recurrence . For the management of the acute phase of the disease, lmwh has largely replaced ufh thus contributing to simplify the management of vte, and now a large proportion of patients with dvt do not need to be hospitalized and can be entirely treated as outpatients . For the long term secondary prevention, vitamin k antagonists remain the only choice for clinicians, and their clear benefits in terms of efficacy need to be periodically balanced in each patient against their risks in terms of safety and their inconvenient management . In a very near future, the armamentarium of clinicians involved in the prevention and treatment of thromboembolic disorders could become much larger . After the positive results of the first clinical trials, new direct thrombin inhibitors and direct factor xa inhibitors that are administered orally are closely approaching the market . With predictable anticoagulant responses and low potential for food - drug and drug - drug interactions these properties and the oral administration render these compounds more convenient than both vitamin k antagonists and lmwh . Based on design of the phase iii clinical trials, we can speculate that some of these compounds will challenge the vitamin k antagonists for the long term secondary prevention of vte, and that other will also challenge the parenteral drugs for the acute phase management, as they are tested as a stand - alone treatment for both dvt and pe . Thus, patients with vte could be treated with a single oral agent right after the objective diagnosis of the disease . Specific areas of particular interest for these new agents include the treatment of patients with cancer and vte, for whom long term treatment with lmwh is currently recommended and for whom an oral agent with a low propensity for drug - drug interactions could represent the ideal therapy, and of course the long term treatment of patients with unprovoked vte, where the complex balance between benefits and risks of the currently available drugs could be simplified with the use of more practical agents.
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This cross - sectional study was conducted on 182 epileptic patients who applied to the neurology polyclinics of the faculty of medicine at erciyes university, kayseri, turkey between november 2011 and november 2012 . Inclusion criteria for the study were established for the participants as follows: to be literate, have no sign of intellectual disability or dementia, have no other disease than epilepsy, be between 18 - 75 years old, have been followed up for epilepsy for at least one year, and to be receiving antiepileptic treatment . The ethics committee of erciyes university, faculty of medicine, approved this study, and informed consent was obtained from the participants . Data collection tools were employed through face - to - face interview with the participants after examinations in a specially allocated area in the neurology polyclinics . As data collection tools, we used the multidimensional scale of perceived social support systems, the beck depression inventory (bdi) and the assessment scale for coping attitudes (cope). This form consisted of 16 questions concerning participants socio - demographical characteristics such as their age, gender, marital status, educational level, occupation, family type, income, and residence . It also covered introductory data regarding their disease such as the duration of the disease, seizure type, the frequency of seizures during the last one year (monthly), duration of disease, the antiepileptic medications taken, how much the disease affected their family and academic life, and how the people around them perceived the disease . The scale was developed by zimmet et al14 in 1988, and the study on its reliability and validity in turkey was conducted by eker et al.15 it is a 7-point likert type scale varying from absolutely no to absolutely yes . It has 3 subscales that are composed of 4 items to assess the sufficiency of support from 3 sources: family, friends, and other important or special individuals . The minimum score to be made on the subscales is 4, while the maximum is 28 . The total scale score is obtained by summing up the scores from the subscales; the minimum score is 4 and the maximum score is 84 . A high score from the scale indicates that the perceived social support is high.4,16 the cronbach - alpha internal consistency coefficient is 0.88, and the reliability coefficient for subscales is 0.90 for perceived social support from friends, 0.83 for perceived social support from family and 0.92 for perceived social support from someone special . The inventory developed by beck et al17 is employed to determine the physical, emotional, and cognitive symptoms of depression . This is a multidimensional coping scale that was developed to assess the responses to stress in different ways.19 it is a self - rating scale that consists of 60 questions and 15 subscales . Each subscale is composed of 4 questions and the possible scores from each subscale range from 4 to 16 . High scores from the scales allow us to interpret which coping attitudes are employed most by the individual . In our study, the cronbach - alpha internal consistency coefficient was found to be 0.79 . The study data was assessed using the ibm spss statistics for windows, version 20.0 . In the study, parametric and non - parametric analyses were conducted after checking the appropriateness of the variables for normal distribution in order to compare the continuous variables . To compare the average scores of 2 groups, we employed the student s t - test and/or mann - whitney u test, while we employed the kruskal - wallis test (kw) or one - way variance analysis to compare the averages of more than one group . Dunn s test, one of the multiple comparison tests (post - hoc), was employed to determine from which group the difference originated . . Multiple linear regression analysis (selection model: stepwise) was conducted to find the independent variables that have an effect using the subtitles of the coping with stress strategies scale . As data collection tools, we used the multidimensional scale of perceived social support systems, the beck depression inventory (bdi) and the assessment scale for coping attitudes (cope). This form consisted of 16 questions concerning participants socio - demographical characteristics such as their age, gender, marital status, educational level, occupation, family type, income, and residence . It also covered introductory data regarding their disease such as the duration of the disease, seizure type, the frequency of seizures during the last one year (monthly), duration of disease, the antiepileptic medications taken, how much the disease affected their family and academic life, and how the people around them perceived the disease . The scale was developed by zimmet et al14 in 1988, and the study on its reliability and validity in turkey was conducted by eker et al.15 it is a 7-point likert type scale varying from absolutely no to absolutely yes . It has 3 subscales that are composed of 4 items to assess the sufficiency of support from 3 sources: family, friends, and other important or special individuals . The minimum score to be made on the subscales is 4, while the maximum is 28 . The total scale score is obtained by summing up the scores from the subscales; the minimum score is 4 and the maximum score is 84 . A high score from the scale indicates that the perceived social support is high.4,16 the cronbach - alpha internal consistency coefficient is 0.88, and the reliability coefficient for subscales is 0.90 for perceived social support from friends, 0.83 for perceived social support from family and 0.92 for perceived social support from someone special . The inventory developed by beck et al17 is employed to determine the physical, emotional, and cognitive symptoms of depression . High total scores indicate a high level and severity of depression . The cronbach - alpha internal consistency of bdi is calculated to be 0.950 . This is a multidimensional coping scale that was developed to assess the responses to stress in different ways.19 it is a self - rating scale that consists of 60 questions and 15 subscales . Each subscale is composed of 4 questions and the possible scores from each subscale range from 4 to 16 . High scores from the scales allow us to interpret which coping attitudes are employed most by the individual . In our study, the cronbach - alpha internal consistency coefficient was found to be 0.79 . The study data was assessed using the ibm spss statistics for windows, version 20.0 . (ibm corp ., armonk, ny, usa). In the study, parametric and non - parametric analyses were conducted after checking the appropriateness of the variables for normal distribution in order to compare the continuous variables . To compare the average scores of 2 groups, we employed the student s t - test and/or mann - whitney u test, while we employed the kruskal - wallis test (kw) or one - way variance analysis to compare the averages of more than one group . Dunn s test, one of the multiple comparison tests (post - hoc), was employed to determine from which group the difference originated . . Multiple linear regression analysis (selection model: stepwise) was conducted to find the independent variables that have an effect using the subtitles of the coping with stress strategies scale . The median age of the 182 participants in the research population was 32 (18 - 75), and 57.7% were women, 47.3% were single, 44% were primary school graduates, 82.45% had a nuclear family, 39% were housewives, 93.4% lived in a city, and 64.8% smoked . It was found that the participants employed emotion - oriented coping strategies most frequently . Among the emotion - oriented coping strategies, religious coping ranked the first, positive reinterpretation and growth came second, and use of an instrumental social support method, which is one of the problem - oriented coping strategies, came third . The mean scores of the male participants were significantly high for positive reinterpretation and growth and use of instrumental social support (p<0.05). When the cope subtitles were compared according to the educational levels of participants, there was a statistically significant difference between the groups for behavioral disengagement and restraint . This difference originated from participants with preschool education and university education for behavioral disengagement, and from participants with university and other educational levels for restraint . Participants with a traditional family structure had significantly lower mean scores for mental disengagement, denial, and religious coping than participants with a nuclear family structure . When the cope subtitles were compared according to occupations, there was a statistically significant difference between the groups for humor, restraint, substance use, and suppression of competing activities . This difference originated from the housewife and other occupations group for humor, and from worker, freelance and other occupations for restraint . Participants who smoked had significantly lower scores than non - smoker participants for humor and behavioral disengagement (p<0.05) (table 2). The seizures of 75.3% of the participants were generalized, and 41.8% had had no seizures during the last month . Seventy - two percent of the participants stated that the response of those around them to their disease was negative, and 73.6% stated that their disease had no effect upon their communication ability . When the cope subtitles were compared according to seizure type, there was a statistically significant difference between the groups for positive reinterpretation and growth, focus on and venting of emotions, active coping, denial, humor, use of emotional social support, acceptance, and planning . This difference originated from the participants who had generalized or partial seizures . When the subtitles of the coping with stress strategies scale were compared according to the frequency of seizures in the last month, the difference was statistically significant for the groups for positive reinterpretation and growth, mental disengagement, denial, religious coping, and use of emotional social support . This difference originated from the participants who had one and more than one seizure a month for positive reinterpretation and growth and use of emotional social support, while for mental disengagement, denial, and religious coping it originated from participants who had more than one seizure a month and those who had no seizures . The mean scores of focus on and venting of emotions were significantly lower for participants who stated that the disease had an effect on their communication ability (p<0.05) (table 2). Our study found a positively significant correlation among the scores of denial, humor, and the duration of the disease there was a positively significant correlation between the scores of planning (table 3). A negatively significant correlation was found among the scores of suppression of competing activities, substance use, use of emotional social support, restraint, religious coping, use of instrumental social support, and the number of friends . While there was a negatively significant correlation between the scores of behavioral disengagement there was a positively significant correlation between the scores of use of instrumental social support and income . There was a positively significant correlation among the scores of humor, behavioral disengagement, and age (p<0.05) (table 3). The correlation between the cope subscale scores of turkish epileptic patients according to different variables . There was a negatively significant correlation among the scores of planning, suppression of competing activities, acceptance, use of emotional social support, religious coping, active coping, use of instrumental social support, focus on and venting of emotions, mental disengagement, positive reinterpretation and growth, and depression (p<0.05) (table 3). We found a negatively significant correlation among the scores of perceived social support from someone special, and in total and positive reinterpretation and growth, focus on and venting of emotions, use of instrumental social support, active coping, restraint, use of emotional social support, acceptance, suppression of competing activities, and planning . There was a negatively significant correlation with the scores of denial and humor (p<0.05) (table 3). As a result of the multiple linear regression analysis, we found that the independent variables affecting the positive reinterpretation and growth subscale of the coping with stress scale were the type of seizures and interference of the disease in communication . The variables affecting the focus on and venting of emotions were the type of seizures and interference of the disease in communication . The variables affecting denial were family structure, smoking, and the type of seizures . The variable affecting the use of emotional social support, acceptance, and planning subscales was the type of seizures (table 4). Multiple linear regression analysis of the factors affecting cope subscales of the turkish epileptic patients . In this study, we found that the participants most frequently employed emotion - oriented coping strategies . Of the emotional coping methods, religious coping ranked first, while positive reinterpretation and growth ranked second . In a study on epileptic patients in iran, it was found that emotion - oriented coping strategies were employed more than problem - oriented coping strategies.21 in their studies, bautista et al22 stated that the most frequently employed basic coping strategies were acceptance, religious tendency, and searching for emotional support, while the less frequently used ones were substance use, denial, and humor . As in numerous studies,21,23,24 our study also suggests that religious belief and intuition are used as a major coping strategy to deal with this chronic disease (epilepsy). Based on these conclusions, the argument related to coping religiously is effective in coping because the spiritual dimension evokes a feeling to accept, to trust in god, and not to rebel by considering the disease as a difficulty originating from allah . However, to come to a final judgment, theist, and atheist groups should be compared . In our study, we found a positively significant correlation among the scores of behavioral disengagement, humor subscales, and age . Bourgault - fagnou and hadjistavropoulos25 reported that the elderly were less sensitive and less anxious regarding their health than young people . This can be explained by the fact that as people age they know their disease better, and they, thus, have fewer negative perceptions about the disease; also their coping capacity and rationalizing ability improve with age . The mean scores of the male participants from positive reinterpretation and growth and use of instrumental social support subscales were significantly higher . It is reported that coping strategies vary according to a variety of factors like age, gender, culture, and disease, and they have distinct characteristics for each individual.20 it is understood from our study that males are more successful in using instrumental social support, which is one of the problem - oriented coping strategies . We believe that this may be connected with attributed power perception, seeking a remedy, and self - expression, to the culture as well . The participants with higher levels of education had significantly lower scores for behavioral disengagement and restraint . Cano et al26 stated that individuals with higher educational levels are more successful and experienced in using the coping strategies against the complex perceptions of feelings of pain . It is not surprising that as the educational level of individuals gets higher, they not only have a better capacity to apprehend the meaning and importance of the disease and its consequences, but also they do not have other tendencies (for example, disengagement and restraint) since they employ solution - oriented coping methods more successfully and consciously . The mean scores of the participants with a traditional family structure were significantly higher than participants with a nuclear family structure for mental disengagement, denial, and religious coping . It has been stated that spirituality has an influence on the coping abilities of participants with their disease.27 religious tendency, and the approaches observed in traditional family structures may lead them to be more effective since they are transferred in a hierarchical order (from generation to generation). We found a positively significant correlation between income and the use of the instrumental social support subscale of cope and a negatively significant correlation with behavioral disengagement . While material income is an advantage in that it provides positive helpful support, since there are more opportunities to look for a remedy, it is understandable that it is also accompanied with a decrease in undermining behavior . As a result of multiple linear regression analysis, seizure type was the independent coefficient that affected positive reinterpretation and growth, focus on and venting of emotions, denial, religious coping, humor, use of emotional social support, acceptance, and planning subscales . We believe that those with generalized seizures are less successful in using coping strategies than those with partial seizures in many parameters, not only due to the severity of the disease but also to perception of the disease as well as to stigmatization, and since they lead to more severe biological outcomes . When the subtitles of the cope scale were compared according to the frequency of seizures during the last month, there was a statistically significant difference between the groups for positive reinterpretation and growth, mental disengagement, denial, religious coping, and use of emotional social support groups . The outcomes produced by epilepsy are reported to make coping with the disease more difficult than seizures do.28 having more seizures adversely affects the coping strategies due to their social - behavioral results, besides the fact that it creates distrust of the treatment provided, a more sick - person mode, and it develops a lack of self - confidence . We found a negatively significant correlation among the number of friends and the scores of using instrumental social support, religious coping, restraint, use of emotional social support, substance use, and suppression of competing activities subscales, while there was a negatively significant correlation with the scores of planning . Participants employ not only positive strategies such as support from family and friends, using distracters, inclining to religion and spirituality, and using meditation techniques, but also negative strategies such as denial, self - accusation, or taking alcohol or drugs.29 people diagnosed with epilepsy experience many psychosocial problems, including the fear of having seizures.30 a life dependent on medications and other people adversely affects self - confidence . Social support and acceptance of the disease by the family play an important role in surmounting such difficulties and social contacts and, within this context, family relations, and social contacts increase a patient s ability to cope with the disease.31 we found a positively significant correlation among the duration of disease and the scores of denial and religious coping subscales of the cope scale . Having a strong faith in religion and an increase in the duration of the disease can explain their having a more submissive attitude in line with their faith and greater acceptance of the reality of the disease . The mean scores of participants who stated that their disease had an influence on their communication ability were significantly lower for the focus on and venting of emotions . The stigma and seizures that accompany epilepsy themselves adversely affect social communication.6 the presence of seizures and their unpredictable times in epileptics may result in social and communicative limitations, loss of functions, and loss of self - confidence . We found a negatively significant correlation among the depression scores, and the scores of positive reinterpretation and growth, mental disengagement, focus on and venting of emotions, use of instrumental social support, active coping, religious coping, use of emotional social support, acceptance, suppression of competing activities, and planning subscales, but we found a positively significant correlation with denial, humor, and substance use . These comorbidity rates are seen in correlation with endogenous and exogenous (iatrogenic conditions) factors and the severity and chronicity of epilepsy.32 depression is obviously common in epilepsy; however, epilepsy is more common in depression . Depression itself is a risk factor for epilepsy.33 lack of family support is a risk factor for depression in people with medical diseases.34 feelings of demoralization, helplessness and hopelessness, confusion, and subjective insufficiency are important for depression, and are determiners of which psychotherapy method should be preferred.6 in a study which investigated the coping strategies developed by patients with epilepsy and depression,22 it was observed that more than one third of the participants with major depression preferred coping through denial . Lower life quality, higher inability, paroxysm of laughter, severe seizures, inability to work and inability to drive was also associated with depression . In general terms, being depressed decreases a person s capacity to cope in many aspects of life, and may lead to a tendency to use drugs because of depression . We found a positively significant correlation among the scores of perceived social support from a friend or someone special, and in total and positive reinterpretation and growth, focus on and venting of emotions, use of instrumental social support, active coping, restraint, use of emotional social support, acceptance, suppression of competing activities, and planning subscales, but we found a negatively significant correlation with denial and humor scores . People with epilepsy are frequently in fear of having a seizure and this affects their social relations, self - confidence, and academic success, and thus leads to making fewer friends, having a shorter married life, and more often developing anti - social behaviour.13 foladi et al3 stressed the greatest need of participants was for emotional support from their family and friends while taleghani et al35 stressed the significance of the presence of a supportive environment in which the problems, experiences, and anxieties of participants are discussed openly . Those with epilepsy are in need of psychological and social support to cope with epilepsy.13 it is obvious from the positive correlation found among the perceived social support and problem - oriented and emotion - oriented coping strategies that social support offers the opportunity to use more conclusive methods and that participants employ more dysfunctional coping methods such as denial when such conditions are not available . A limitation of this study was the use of the bdi as the only assessment tool to assess the presence and severity of depressive symptoms . In conclusion, this study found that people with epilepsy employed emotion - oriented coping strategies most frequently . The most important variables affecting coping strategies with the disease among epileptics were age, gender, educational level, family structure, seizure type, and interference of the disease in the patient s communication ability . It is crucial for epileptics to keep losses related to their disease to a minimum by not only controlling the frequency and severity of seizures through optimal effective medication, but also by developing social support systems, taking the necessary precautions to protect their mental health and treating existing mental problems, if there are any.
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Slipped capital femoral epiphysis (scfe) is an adolescent hip disorder with displacement of the capital femoral epiphysis from the metaphysis through the physis . Most scfes are varus (medial and posterior epiphyseal displacement relative to the metaphysic) but can occasionally be valgus (lateral and superior epiphyseal displacement) [14]. The vast majority are idiopathic; atypical scfes are those that occur due to an endocrine disorder [57], renal failure osteodystrophy, or radiation therapy [7, 9, 10]. The literature regarding the epidemiology and demographics of scfe first requires a general knowledge of the disorder . The traditional clinical classification was acute, chronic, and acute on chronic [1216], based on the patient's history, physical examination, and roentgenograms . An acute scfe is defined as those with symptoms for <3 weeks with an abrupt displacement through the proximal physis . Acute - on - chronic scfes are those with chronic symptoms initially and the subsequent development of acute symptoms . Newer classifications are more clinically useful, depend upon physeal stability, and predict the prognosis regarding subsequent avascular necrosis . A stable scfe is defined as one where the child is able to ambulate, with or without crutches . An unstable scfe is defined as one where the child cannot ambulate, with or without crutches . The radiographic classification depends upon the presence or absence of a hip effusion on ultrasonography [19, 20]. The absence of metaphyseal remodeling and the presence of an effusion define an unstable scfe; metaphyseal remodeling and the absence of an effusion define a stable scfe . Unstable scfes have a much higher incidence of avascular necrosis (up to 50% in some series) compared to stable scfes (nearly 0%). Radiographs demonstrate an inferior and posterior slip of the proximal femoral epiphysis relative to the metaphysis . The gradual slip demonstrates radiographic signs of remodeling on the superior and anterior femoral metaphysis, and periosteal new - bone formation at the epiphyseal - metaphyseal junction posteriorly and inferiorly . In the early slip as such it is usually seen early only on the lateral view, and both anteroposterior and lateral radiographs must be obtained . Other radiographic signs of an early scfe are the metaphyseal blanch sign of steel and klein's line . The metaphyseal blanch sign of steel is a radiographic double density seen on the anteroposterior view at the level of the metaphysis; this double density reflects the posterior cortical lip of the epiphysis as it is beginning to slip posteriorly and is radiographically superimposed upon the metaphyseal density . Klein's line is drawn along the anterior or superior aspect of the femoral neck; the epiphysis should normally intersect this line . In an early scfe the epiphysis will be flush with or even below this line (figure 1). The first is the amount of displacement of the epiphysis on the metaphysis (figure 2). A mild scfe is defined as epiphyseal - metaphyseal displacement <1/3 the width of the metaphysis; a moderate scfe 1/31/2; a severe scfe> 1/2 (figure 2(a)). This method is less accurate than angular measurement since distinct landmarks are difficult to determine due to metaphyseal remodeling in the gradual stable scfe . Angular measurement uses the epiphyseal - shaft angle on the frog - lateral radiograph (figure 2(b)) and is categorized into 3 groups: mild <30, moderate 3050, and severe> this classification is important for long - term prognosis as mild and moderate slips have a much better long - term prognosis than severe slips which demonstrate a more rapid development of degenerative hip disease [26, 27]. Scfe has been known by many different names since its first description in the late 19th and early 20th centuries . Prior to the last 40 years or so, even now there is no actual mesh (medical subject heading) term for slipped capital femoral epiphysis (scfe). Slipped capital femoral epiphysis or scfe, must be searched as a keyword phrase . To ensure capture of all the published literature, older terms were also searched as keywords or keyword phrases . Therefore the terms used to search for scfe the last strategy was to combine these two sets of terms: (slipped or slipping or sliding or displaced or displacement or detached or detachment or separated or separation) combined with (femur or head or capitis or epiphysis or epiphyseal or epiphysiolysis or epiphysiolyses). The databases searched were pubmed (19472010) (http://www.ncbi.nlm.nih.gov/pubmed/), ovid medline (19472010), embase (19472010), worldcat (18802010) (books and theses) (http://firstsearch.oclc.org/), google scholar (18802010) (http://scholar.google.com/), web of knowledge (19872010), and indexcat (18801961) (index catalogue of the library of the surgeon - general's office) (http://www.indexcat.nlm.nih.gov/). Exclusion criteria were those manuscripts discussing surgery, therapy, rehabilitation, and any foreign language articles without an english abstract . Individual orthopaedic journals were also searched for articles published prior to 1966 that predate electronic medline indexing, including journal of bone and joint surgery (american and british), clinical orthopaedics and related research, and acta orthopaedica scandinavica . Age groups were limited to those <18 years old; duplicate citations were removed . These 1407 manuscripts were reviewed to find those that discussed any of the topics regarding scfe and epidemiology, demographics, incidence, prevalence, race, gender, family history, inheritance, genetics, age, bone age, weight (either birth weight or normal weight), height, growth, maturation, any other anthropometric characteristics, seasonal variation, hormone, endocrine, congenital anomalies, collagen, immunoglobulin, and opposite hip . Of these 1407 manuscripts, 114 provided ample information and are the contents of this paper . Conventional quotation for scfe incidence in the literature is the number of cases per 100,000 (usually for the age range 8 to 15 years old, although some use <25 years of age) and is used throughout this paper . The incidence (table 1) ranges from 0.2 per 100,000 in eastern japan to 17.15 in the northeastern united states . Recent studies indicate that the overall incidence in the united states is 10.8/100,000, similar to 10.08 in boys in the kelsey et al . 's study . In japan it has increased to 2.22/100,000 for boys and 0.76/100,000 for girls . In olmsted county, minnesota, the incidence is increasing in some areas compared to previous reports; in new mexico it has nearly tripled (6.05 versus 2.13) compared to an earlier study . In scotland, the incidence has increased from 3.78 in 1981 to 9.66 in 2000, correlated with rising obesity . The incidence of scfe in southern sweden increased in rural areas for boys (7.5 versus 4.8) but decreased for girls (2.2 versus 3.8). No differences were noted in connecticut in white children between urban and nonurban settings (3.19 for both) although there was an increased incidence in africans living in urban areas compared to nonurban areas (7.95 versus 1.35). There are differences by geographic region . In a recent study the highest incidence was in the northeast at 17.15 and the lowest in the midwest at 7.69 . This is similar to an earlier study where the incidence in the northeast was 10.08 and 2.13 in new mexico . Race is classified using the definitions of eveleth and tanner: caucasians, africans in africa and of african ancestry, asiatics (amerindians, hispanics, indonesian - malays), indo - mediterraneans (inhabitants of the near east, north africa, and indian subcontinent), and australian aborigines and pacific island peoples . The relative racial frequency (rrf) of scfe, with whites at 1.0, is 4.5 for pacific islanders, 2.2 for africans, 1.05 for amerindian (native americans and hispanics), 0.5 for indonesian - malay (chinese, japanese, thai, vietnamese, etc . ), and 0.1 for indo - mediterranean peoples (near east, north african, or indian subcontinent ancestry). More recent data indicates that these numbers, relative to caucasians, is 5.6 for polynesians, 3.9 for blacks, and 2.5 for hispanics (figure 3(a)) [29, 31]. In new zealand the rrf was 5.6 for the maori peoples and 4.2 for other pacific islanders . In singapore the rrfs, relative to chinese, are 9.6 for indo - mediterraneans (indian), 2.8 for malay, and 3.0 for mixed (eurasians) (figure 3(b)). Thus indo - mediterraneans (indian) had a 9-fold increased incidence compared to indo - malay (chinese). This is in contrast to another study where indo - mediterraneans had a 5 times lower frequency of scfe compared to indo - malays (all groups these racial differences most likely reflect the average body weights for each racial group and further support the significant role that obesity and mechanical stress plays in the etiology of scfe . A less likely explanation is racial variability in acetabular depth and femoral head coverage; the acetabulae in african children were deeper than white children in one study, but not in another study . Early in this century 90% occurred in males but has now decreased to ~60% . In a review of 4343 children, there are differences in gender by race, with indo - mediterraneans having the highest proportion of boys (90%) and polynesians an equal male / female ratio . Early in this century the average age was much higher with a gradual decrease over time (figure 4(a)). Fifteen years ago the average age was 13.5 years for boys and 12.0 years for girls and is now 12.0 years for boys and 11.2 years for girls (table 3). In scotland, the age has dropped from 13.4 to 12.6 years over a 20-year period . One study demonstrated different ages at presentation by racial group (figure 4(c)). The physiologic age range during which scfe occurs is less variable than the chronologic age range (figure 5), termed the narrow window [56, 63]. The average symptom duration for stable scfes is 4 to 5 months (with minimal difference by gender). In 2482 children, the average symptom duration was 4.3 months; 4.5 months for boys and 3.6 months for girls (table 4). Two recent studies have noted a decrease in symptom duration to 2 - 3 months [46, 88]. Although there is a general correlation between symptom duration and scfe severity there is considerable variability (figure 6(a)). For any given individual child, slip severity and symptom duration is unique; in a large population, there is a weak positive correlation with slip severity and symptom duration . Mild scfes have a shorter duration of symptoms than severe scfes (figure 6(b)) which suggests that all scfes start at a similar age; those children with severe scfes just had a longer symptom duration and time to presentation than those with milder scfes . In a review of 328 stable scfes this was documented; a child with a stable scfe was 2.0 times more likely to have a moderate or severe scfe if> than 12.5 years at diagnosis than if <12.5 years at diagnosis, and a child with a stable scfe is 4.1 times more likely to have a moderate or severe scfe if the duration of symptoms is> than 2 months than if 2 months . Other positive predictors of increasing time to diagnosis are knee / distal thigh pain, medicaid coverage, lower family income, and a stable scfe . Slip severity and symptom duration is greater in children presenting with knee pain compared to other symptoms [8891]. In a study of 45 children, 15 presented solely with knee pain having an average symptom duration of 8.3 months; 74% of the scfes were severe . The remaining 30 presented with other symptoms with an average symptom duration of 6.5 months; 24% of the scfes were severe . Reviewed 87 children with stable scfes, 20 had a delayed diagnosis, with 8 (40%) being severe . In leeds, england, those children with scfe diagnosed in the emergency department compared to general practitioners had a shorter symptom duration (95 versus 119 days) and fewer severe scfes (8 versus 21%). One study, however, did not see a difference in symptom duration in scfe children where the diagnosis had been initially missed due to the absence of hip pain (average symptom duration 127 days and 146 days in those whose diagnosis was and was not missed). It would seem reasonable that as symptom duration increases radiographic changes should concomitantly progress (more metaphyseal remodeling). The presence or absence of a positive klein line and metaphyseal remodeling (superior and anterior smoothing, inferior and posterior buttressing) were directly correlated to symptom duration but with significant variability . Those with more metaphyseal changes had higher bmis, interpreted as the larger body mass per unit height resulting in more bony reaction / adaptation according to wolf's law . The age at diagnosis of the 1st scfe correlates with scfe severity and is also related to the narrow physiologic window [56, 63]. Subtracting the average symptom duration for the mild, moderate, and severe categories from the average age at presentation results in a similar average age at onset (figure 6(c)), confirming the narrow bone age window . 50% of children with scfe are> 95th percentile weight for age [30, 9496]. Age at diagnosis decreases with increasing obesity; 12.4 years for those over the 95th percentile weight for age and 14.3 years for those under the 10th percentile weight for age . Recently body mass index (bmi) [97, 98] has been used to evaluate body habitus; the average bmi in scfe children is 2530 kg / m or> 85th percentile [39, 46, 99102]. The average bmi of children with bilateral scfe is higher than with unilateral scfe (26.8 versus 31.1 kg / m). Obese children have decreased femoral anteversion, and scfe children have femoral retroversion even in the noninvolved hip [103, 104]. This, along with biomechanical studies demonstrating an increased physeal stress in retroverted hips and the many demographic studies associating obesity with scfe, supports the theory that obesity is intimately involved in the development of most idiopathic scfes . In latitudes north of 40 scfe presents more frequently in the late summer and autumn months (figure 7(a)) [34, 58, 59, 61, 62, 106, 107]. Subtracting symptom duration from the time of presentation demonstrates that the scfe onset is in the early summer / late spring months (figure 7(b)). The reported proportion of bilaterality depends upon the study, method of radiographic measurement, race, and treatment (table 5). Most series report 18 to 50% bilaterality with recent follow - up studies into adulthood quoting 63% [34, 109, 110] and 66% . Bilaterality is more frequent in africans (34%) than hispanics (17%), whites (17%), or asians (18%). Treatment may affect risk of bilaterality; bilaterality is 36% with in situ fixation and 7% spica cast treatment . Close attention is mandatory to the opposite normal hip in those children with a unilateral scfe treated by in situ fixation . The age at presentation is younger in children who present with a unilateral scfe that later develops bilateral scfes compared to those who do not develop bilateral scfes [17, 50, 59, 113116]. This age difference is seen in both chronologic age (12 versus 13 years of age) [17, 30, 115, 116] and bone age . In one study the overall difference in chronologic age in those with a unilateral scfe that became bilateral compared to those that stayed unilateral was 1 year (12 versus 13 years); in another study 0.9 years for girls (11.0 versus 11.9 years) and 2.2 years (12.1 versus 14.2 years) for boys; and in a 3rd study 11.5 versus 12.7 years, with those children aging <12 years having a unilateral scfe demonstrated a 3.8 times increased risk of developing a contralateral scfe . In a study of 50 children with unilateral scfe using a modified oxford hip bone age, a contralateral scfe occurred in 85% of hips having a score of 16, 11% of hips having a score of 21, and never when the score was 22 . Once the triradiate cartilage has closed, the risk of a contralateral scfe is only 4%, although another study did not find any association between the status of the triradiate cartilage, other skeletal maturity markers, and subsequent bilaterality . The risk of bilaterality is increased when the posterior slope angle of the capital femoral epiphysis is higher [119, 120]. In children with bilateral scfes, 5060% present with simultaneous bilateral involvement . In those children with sequential bilateral scfes, 8090% of the second scfes occur within the first 18 months after the first scfe (figure 8) [17, 29, 30, 34, 50, 67, 102, 121, 122]. Tests for a disease are evaluated in terms of sensitivity, specificity, positive predictive value, and negative predictive value . Sensitivity is the proportion of individuals in a tested population who actually have the disease and are identified as having it with the test . Specificity is the proportion of individuals in a tested population who do not have a given disease and are identified as not having it with the test . The probability that a person with a positive result truly has the disease is the positive predictive value; the probability that a person with a negative test truly does not have the disease is the negative predictive value . When evaluating a child with a newly diagnosed scfe, the underlying etiology immediately comes into consideration . This is for both diagnostic concerns with potential medical issues as well as orthopaedic treatment . The underlying medical issues involve significant anesthetic concerns; treatment involves the question of prophylactic fixation of the opposite hip when the child presents with a unilateral scfe . Any test that can give an accurate negative predictive value for a child with a scfe not having an atypical scfe is important . If the test has a high negative predictive value, the clinician can be confident that the scfe is idiopathic . The history of prior radiation therapy is usually discovered in the initial evaluation . However, many times it is the orthopaedic surgeon who picks up on an underlying medical problem as the etiology of the scfe (e.g. Endocrine dysfunction, renal osteodystrophy). In 2001 the age - weight test was described to assist in the differentiation between an idiopathic and atypical scfe . The age - weight test is defined as negative when age <16 years and weight 50th percentile and positive when beyond these boundaries (table 6). Is the child having an atypical scfe, absence of disease is the child having an idiopathic scfe . The demographics of 433 children (285 idiopathic, 148 atypical) with 612 scfes were studied to define predictors of atypical scfes . There was significant variability in both age and bilaterality between the atypical and idiopathic groups (figure 9). Multiple logistic regression analysis demonstrated that age and weight were predictors of an atypical scfe . For two patients of equal weight, those <10, or> 16 years of age are 4.2 times more likely to have an atypical scfe; for two patients of equal age, those <50th percentile weight are 8.4 times more likely . The probability of a child with a negative test having an idiopathic scfe is 93%, and a child with positive test having an atypical scfe is 52% . In the same year . Showed that the height at diagnosis was an important predictor of an endocrinopathy . The sensitivity and negative predictive value of detecting an underlying endocrinopathy in a patient with a scfe who was <10th percentile in height for age were 90.2% and 98% . The age - weight test was further confirmed in 2006, along with the definition of the height and age - height tests . The age - height test was defined using the same cells as the age - weight test except that the percentiles applied to height rather than weight . The height test is defined as positive if the child's height is 10th percentile and negative if> 10th percentile for age . The height test has a positive and negative predictive value of 75% and 97% . Of these three tests, the height test is likely to be the most useful in the differentiation between a typical and atypical scfe if the height of a child can be obtained . When the height cannot be obtained, the age weight test will result in a similar negative predictive value but with a lower positive predictive value . Thus the weight, and where possible, the height of any child newly diagnosed with a scfe should be obtained to assist in the differentiation between an atypical and idiopathic scfe . Familial scfe was first noted in the english language literature in 1940 with many more subsequent descriptions [6574, 7680, 8387]. Proposed patterns of genetic transmission include x - linked dominant, autosomal dominant with variable penetrance, and autosomal recessive [68, 71, 73, 77]. Hla phenotype studies in children demonstrate no common findings (table 7) [74, 75, 7982, 86]. Other skeletal abnormalitiesin most children with scfe, femoral anteversion is decreased . In a 3-dimensional ct scan study of 30 scfes there was a reduction in femoral anteversion from 12.7 to 7.0 as well as a reduction in the femoral neck shaft angle from 141 to 134. in another ct scan study of 25 scfes a reduction of femoral version 1 was noted in the involved hip and 7 in the uninvolved hip in unilateral cases . In a 3rd ct scan study of 25 scfes femoral anteversion was 9.8 and 25 in a control group of children between the ages of 8 and 16 years . Patients with acute scfes have less reduction in anteversion compared to those with chronic scfes (9.3 versus 0.7) in the involved hip and uninvolved hip (15.7 versus 11.8), with an average difference in version in acute scfes of 6.7 and in chronic scfes of 13.9 . There are also no abnormalities in tibial torsion in children with stable scfes .genu recurvatum has been fleetingly described in scfe [132, 133]. It is believed that this represents a gradual anterior slip of the proximal tibial epiphysis, with the physes perhaps universally weak or susceptible to large stresses from obesity . Surprisingly, tibia vara (blount's disease) is very rarely associated with scfe [134136] even though both conditions typically occur in obese children . Peroneal spastic flatfoot has been associated in certain cases with scfe .increase width of the symphysis pubis and unaffected hip joint cartilage space has been observed, suggesting an abnormality of cartilage formation or maturation on a macroscopic level rather than a microscopic level . Skewness of the symphyseal joint has also been described on standing radiographs of the pelvis . In most children with scfe, femoral anteversion is decreased . In a 3-dimensional ct scan study of 30 scfes there was a reduction in femoral anteversion from 12.7 to 7.0 as well as a reduction in the femoral neck shaft angle from 141 to 134. in another ct scan study of 25 scfes a reduction of femoral version 1 was noted in the involved hip and 7 in the uninvolved hip in unilateral cases . In a 3rd ct scan study of 25 scfes femoral anteversion was 9.8 and 25 in a control group of children between the ages of 8 and 16 years . Patients with acute scfes have less reduction in anteversion compared to those with chronic scfes (9.3 versus 0.7) in the involved hip and uninvolved hip (15.7 versus 11.8), with an average difference in version in acute scfes of 6.7 and in chronic scfes of 13.9 . It is believed that this represents a gradual anterior slip of the proximal tibial epiphysis, with the physes perhaps universally weak or susceptible to large stresses from obesity . Surprisingly, tibia vara (blount's disease) is very rarely associated with scfe [134136] even though both conditions typically occur in obese children . Increase width of the symphysis pubis and unaffected hip joint cartilage space has been observed, suggesting an abnormality of cartilage formation or maturation on a macroscopic level rather than a microscopic level . Skewness of the symphyseal joint has also been described on standing radiographs of the pelvis . Other nonskeletal associationsimmune complexes in the synovial fluid and synovium of the hip, but not in the serum, have been found in a high percentage of scfe cases [140, 141]. This is different from the synovitis of the knee or hip caused by other disorders where immune complexes are rarely found . Fluorides in the water, used to reduce dental caries, also strengthen the physes and metaphyseal bone; however, no difference in the incidence of scfe was seen in children with or without fluoride exposure . Immune complexes in the synovial fluid and synovium of the hip, but not in the serum, have been found in a high percentage of scfe cases [140, 141]. This is different from the synovitis of the knee or hip caused by other disorders where immune complexes are rarely found . Fluorides in the water, used to reduce dental caries, also strengthen the physes and metaphyseal bone; however, no difference in the incidence of scfe was seen in children with or without fluoride exposure . The current incidence of scfe ranges from 0.33/100,000 to 24.58/100,000 children 8 to 15 years of age, depending upon gender and ethnicity . There is significant variability within racial groups and the relative frequency, relative to caucasians at 1.0, is 5.6 for polynesians, 3.9 for blacks, and 2.5 for hispanics . The average age is 12.0 years for boys and 11.2 years for girls; obese children present earlier than light weighted children . The physiologic age range during which scfe occurs is less variable than the chronologic age range . Although there is a general correlation between symptom duration and scfe severity, there is considerable variability . The majority of children are obese with> 50% of children> 95th percentile weight for age; the average bmi in scfe children is 2530 kg / m or> 85th percentile . Bilaterality ranges from 18 to 50% and is more frequent in africans compared to hispanic, white, and indo - malay peoples . In children with bilateral involvement, 5060% present with simultaneous scfes; 8090% of those who present with a unilateral scfe and the age at presentation is younger for those who present with a unilateral scfe and develop contralateral involvement compared to those who do not develop a contralateral scfe . The age - weight, age - height, and height test are useful to differentiate between an idiopathic and atypical scfe.
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Moderate to severe gastroesophageal reflux in neonates and infants is often a complex interaction of developmental and anatomic conditions that limits adequate enteral intake and may be associated with significant morbidity . Common comorbidities in infants with gastroesophageal reflux include failure to thrive, prematurity, neurological impairment, and chronic respiratory conditions, all of which can be exacerbated by anatomic anomalies that further predispose to the development or progression of gastroesophageal reflux . Although a laparotomy will often correct a congenital or acquired anatomic anomaly, the surgical adhesions that form as a result of a laparotomy may complicate subsequent surgical therapy for gastroesophageal reflux . Many studies document the safety and long - term efficacy of laparoscopic nissen fundoplication for the treatment of gastroesophageal reflux in infants and children . The laparoscopic approach is feasible as a primary procedure and a secondary procedure after failure of a previous open or laparoscopic reflux operation . However, few studies have examined the efficacy of laparoscopic nissen fundoplication in infants and children after previous laparotomy for diseases or problems unrelated to reflux . The purpose of this study is to examine our experience with open and laparoscopic nissen fundoplication in a population of infants with a history of previous neonatal laparotomy for diseases unrelated to gastroesophageal reflux . An institutional review board - approved retrospective review (irb #05091500) of our surgical procedure database at the children's hospital of pittsburgh was conducted to identify all infants with a history of neonatal laparotomy who subsequently required surgical therapy for gastroesophageal reflux . Study dates were january 1, 2000 through september 1, 2005 . During the period reviewed, 32 infants were identified with a history of neonatal laparotomy and subsequent surgical correction of gastroesophageal reflux . Only infants requiring surgical correction of reflux within the first year of life, and therefore less than 12 months after neonatal laparotomy, were included in this study . This 12-month time frame was selected because we specifically chose to evaluate infants, and it represented a more homogeneous subset of patients when comparing laparoscopic nissen fundoplication (lnf) and open nissen fundoplication (onf) groups . The 6 excluded patients were all older than 12 months at the time of nissen fundoplication (3 in each group, age range 2 to 15 years old). These 26 infants underwent laparotomy during the neonatal period for multiple different diagnoses including intestinal atresia (duodenal, jejunal and ileal); necrotizing enterocolitis; meconium ileus; gastroschisis; omphalocele; malrotation; congenital diaphragmatic hernia and imperforate anus . Twelve infants underwent lnf and 14 infants underwent onf, thereby forming the 2 study groups . Gastroesophageal reflux was identified clinically, radiographically, or with both methods, and the infants underwent surgical correction of the reflux at the discretion of the attending surgeon . All infants had either a barium esophagram or a nuclear gastric emptying scan, which documented gastroesophageal reflux before anti - reflux surgery . A nissen fundoplication was performed in all cases either open or laparoscopically by dividing the short - gastric vessels and creating a 360-degree wrap of the fundus around the esophagus . Ten of 12 infants in the lnf group had concomitant feeding gastrostomy tubes placed laparoscopically . Two infants in the lnf group already had gastrostomy tubes in place at the time of fundoplication, both of which were placed by the open method, one on the first day (duodenal atresia) and the other at one month of age (jejunal atresia). In the first infant, the tube was taken down laparoscopically at the time of fundoplication and replaced in a new position on the gastric wall to accomplish the wrap . In the second infant, thirteen of 14 infants in the onf group had a feeding gastrostomy tube placed at the time of fundoplication with 3 of these infants having relocation of an existing gastrostomy tube . Outcome variables included age at initial laparotomy, number of previous laparotomies before surgical correction of the gastroesophageal reflux, age at the time of fundoplication, weight at the time of fundoplication, operative time for fundoplication, length of stay, and time to resumption of enteral feeds after fundoplication, recurrence of gastroesophageal re - flux, and mean follow - up duration . In general, infants with these neonatal diagnoses, with or without antireflux surgery, are followed for a minimum of 5 years by our group . Of the 26 infants identified with a history of neonatal laparotomy and subsequent surgical correction of gastroesophageal reflux within 12 months of life, 12 infants underwent laparoscopic nissen fundoplication (lnf), and 14 infants underwent open nissen fundoplication (onf). In the lnf group, diagnoses included 4 neonates with gastroschisis, 3 neonates with intestinal atresia (1 duodenal, 1 ileal, 1 jejunal), 2 neonates with meconium ileus (both underwent ileostomy and mucus fistula at day of life #1 with re - anastomosis at 2 to 3 months of age), and 1 each with omphalocele, malrotation, and perforated necrotizing enterocolitis . In the onf group, diagnoses included 4 neonates with congenital diaphragmatic hernia (all left - sided, repaired by laparotomy), 4 neonates with gastroschisis, 2 neonates with perforated necrotizing enterocolitis, 2 neonates with imperforate anus / tracheoesophageal fistula, 1 infant with omphalocele, and 1 infant with intestinal atresia . In the lnf group, 4 infants had intestinal stomas performed at initial operation with subsequent closure before fundoplication . In the onf group, 3 infants had stomas created at initial laparotomy and subsequent takedown before fundoplication . There was no difference between the 2 groups relative to age at the time of initial laparotomy (1 day of life for lnf vs 14 days of life for onf, p=0.08). What may appear as a large difference in days of life at initial operation may be accounted for by the fact that 2 patients in the onf group had initial laparotomies at ages 51 days (ileostomy/ mucus fistula for perforated nec in ex-25 week gestation infant) and 90 days (duodenoduodenostomy for duodenal atresia in 1.5-kg infant born at 800 g). There was no difference in age at the time of the fundoplication (5.5 months for lnf vs 6.6 months for onf, p=0.57), nor was there any difference between the 2 groups with respect to infants' weight at the time of the fundoplication (5.2 kg lnf vs 5.7 kg onf, p=0.51). The number of previous laparotomies prior to fundoplication was similar in each group (1.7 laparotomies for lnf vs 1.9 laparotomies for onf, p=0.36). Additionally, operative time was comparable between the groups with an average operative time of 131 minutes in the lnf group (range, 77 to 190) and 164 minutes in the onf group (range, 100 to 287) (p=0.10). Infants undergoing lnf did resume enteral feeds earlier than those undergoing onf (3.0 days lnf vs 6.3 days onf, p=0.01). The length of stay after fundoplication was actually shorter in the lnf group (14 days lnf vs 26 days onf, p=0.13), but this did not reach statistical significance . All infants in both groups progressed to tolerate full oral, gastrostomy, or both, feeds with appropriate weight gain during follow - up periods . Subjectively, no infants in either group developed recurrent gastroesophageal reflux or wrap failure during an average follow - up period of 22 months (range, 1 to 54). Follow - up length of time for the lnf group was not different from that in the onf group (1 to 28 months versus 1 to 54 months, respectively, p = ns). However, routine monitoring for recurrent reflux is not done at our institution unless an infant develops feeding intolerance after fundoplication . Our results suggest that laparoscopic nissen fundoplication (lnf) is at least as safe and effective as open nissen fundoplication (onf) in the surgical treatment of gastroesophageal reflux in a population of infants with a history of a neonatal laparotomy . Additionally, lnf appears to have a distinct advantage by allowing an early resumption of full enteral feeds after nissen fundoplication . All of the infants in this study required an operation in the neonatal period for surgical diseases unrelated to gastroesophageal reflux . Both the onf and the lnf groups included infants with a variety of neonatal surgical diseases, many of which predispose to the subsequent development of gastroesophageal reflux . Specifically, there is a well - documented increase in the incidence of gastroesophageal reflux in neonates with a history of gastroschisis, congenital diaphragmatic hernia, and omphalocele . The risk for the development of gastroesophageal reflux in these infants is theorized to be secondary to defects of bowel motility, increased abdominal pressure, and gastroesophageal junction anatomical anomalies . At least half of the infants in both the onf and lnf groups had a variety of abdominal wall and diaphragmatic defects . There have been few studies in infants and children that address the safety and efficacy of laparoscopic nissen fundoplication for the treatment of gastroesophageal re - flux in children with previous abdominal surgery . Best studied is the role of laparoscopic nissen fundoplication for recurrent gastroesophageal reflux after either an open or laparoscopic primary fundoplication . Rothenbergs has one of the largest series of laparoscopic redo nissen fundoplications with 118 patients ages 6 months to 19 years . In this study, redo laparoscopic nissen fundoplication was safe and efficacious in these infants, children, and adolescents . Other authors have also documented the safety and efficacy of laparoscopic redo nissen fundoplications in infants and children of a variety of ages . However, fewer studies have examined the role of laparoscopic nissen fundoplication after neonatal laparotomy for diseases unrelated to gastroesophageal reflux . In a study by liu et al, the authors concluded that laparoscopic nissen fundoplication was feasible and safe in a population of children with previous ventriculoperitoneal shunt placement, gastrostomy placement, or both . Similarly, lintula et al15 concluded that a pre - existing gastrostomy tube did not preclude a safe and effective laparoscopic nissen fundoplication in children . Van der zee et al both documented the technical feasibility and safety of nissen fundoplication in children with a pre - existing gastrostomy tube . Although these studies do examine redo laparoscopic surgery, none of these studies have examined the role of a laparotomy for primary neonatal surgical disease pre - existing the development of gastroesophageal reflux . There are distinct nuances in achieving safe minimally invasive access in infants after neonatal laparotomy . For infants with abdominal wall defects (eg, gastroschisis and omphalocele), the umbilicus is avoided because the majority of these infants have dense adhesions in this area from prior repair . In our study, initial access for these patients was achieved through a left upper quadrant cut down approach with a 4-mm port . In patients with congenital diaphragmatic hernia and intestinal atresia, adhesions from a prior laparotomy thus, access via the umbilicus was attempted and successful for these patients . For some patients, extended operative times were due to optimizing port placement and extensive adhesiolysis to complete a laparoscopic nissen fundoplication . Unfortunately, the actual times for this aspect of the study operations were not recorded separately, and therefore, we are unable to determine the exact time for doing the fundoplication portion of the operation . Beyond the technical feasibility of a laparoscopic nissen fundoplication at any time point after a neonatal laparotomy, our study also highlights the feasibility of a laparoscopic nissen fundoplication in a group of very small infants . Specifically, all of the infants in the lnf group underwent fundoplication at an average weight of 5.2 kg (range, 2.7 to 8.8). These infants were, on average, smaller than the infants and children in the largest series of redo laparoscopic nissen fundoplication, in which the lowest documented weight was 6.4 kg . However, we are not aware of any studies that have examined weight as a selection criterion for redo laparoscopic nissen fundoplication . The study dates include a period of transition in our institution with respect to surgical approaches to gastroesophageal reflux . In 2001, an experienced laparoscopic surgeon joined the surgical group, resulting in a gradual conversion over to advanced laparoscopic approaches to historically open surgical diseases . The number as well as the approach for surgical anti - reflux operations at our institution increased and changed over the time period of this study from 41 in 2000 (37 onf; 4 lnf) to 122 in 2005 (118 lnf; 4 onf). As advanced laparoscopic skills have developed in the group, the operative times for primary lnf have ranged from 45 minutes to 135 minutes, depending on staff and resident trainee experience . More surgeons will now consider primary laparoscopic nissen fundoplication, even in the face of previous abdominal surgery . We intend to review our experience for all anti - reflux operations in the neonatal age group or in infants under 7 kg to determine the outcomes and benefits of such an approach in this patient population . Currently, a primary laparoscopic approach is our preferred approach to gastroesophageal reflux, regardless of the history of previous operations . Outcomes following laparoscopic or open nissen fundoplication performed in infants after a neonatal laparotomy are similar . However, a laparoscopic fundoplication did allow for earlier return to enteral feeds compared with the open approach and tends toward shorter hospital length of stay . Therefore, we conclude that laparoscopic nissen fundoplication is technically feasible, safe, and effective in the treatment of gastroesophageal reflux in infants with a previous neonatal laparotomy.
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Any decision - making process in clinical medicine may be simplified in a series of sequential steps that cumulatively lead to a clinical diagnosis or intervention . Thus, the additional benefit of any step must be balanced with the associated exploitation of medical and economic resources . When limiting our focus to diagnostic process, the additional value of any subsequent test will depend on the absolute increase in accuracy as compared with what was achieved prior to the test . Histological examination of colorectal lesions has been considered in the last decades as a critical step in the decision - making process, because of its contribution in differentiating between nonneoplastic and neoplastic lesions, and - among those neoplastic - between noninvasive and invasive lesions.1,2 the relevance of postpolypectomy histological examination has been intimately related with the lack of any alternative in characterizing polypoid or nonpolypoid lesions prior to the pathological examination itself.1,2 despite histological characterization of colorectal polyps has been generally considered as a gold - standard, some pitfalls may be identified . First, not all the removed polyps are actually retrieved, resulting into a complete loss of information in 10% to 20% of cases.3 secondly, a high interobserver variability in the diagnosis of the degree of dysplasia and villous component - i.e., the unfavourable features discriminating between advanced and nonadvanced neoplasia - has been shown.4 third, the same differentiation between hyperplastic and adenomatous histotypes is not perfect, with adenomatous tissue hidden in the nonexamined part of the embedded specimen . Fourth, there is no general consensus on the clinical meaning of histological characterization with divergent recommendations according to different guidelines.5,6 the need of postpolypectomy histological examination causes a substantial exploitation of medical and economic resources . It may be estimated that roughly one in every two patients undergoing screening or diagnostic colonoscopy are diagnosed with at least one polyp, thus requiring postpolypectomy pathological examination.7,8 secondly, the vast majority of these polyps are diminutive (5 mm) with a very low prevalence of advanced neoplasia, marginalizing the clinical impact of pathological examination.7 - 9 third, the estimated cost of the pathological examination is of the same magnitude with that of colonoscopy, heavily affecting the cost of colonoscopy screening without adding an equivalent contribution to colonoscopy efficacy.10 - 12 fourth, the need of a new visit for communicating the postpathological surveillance interval generates a substantial and avoidable loss of productivity for the society, especially when considering that roughly 60% of the united states population undertakes a screening colonoscopy every 10 years.13 in order to address these pitfalls, a policy of discarding the postpolypectomy pathological examination has been proposed, also named as predict - resect - and - discard policy.14 - 16 its main assumption is that if endoscopy may predict in vivo the histotype of the polyp, the additional value of histological examination may be marginalized, becoming inefficient and/or cost - ineffective.10 - 12 predict - resect - and - discard policy has been mainly prompted by the technical evolution of endoscopic imaging, leading to a new field defined as advanced endoscopic imaging, with the main aim of differentiating between hyperplastic and neoplastic, as well as between noninvasive and invasive neoplastic lesions.17,18 however, before implementing a predict - resect - and - discard policies, several issues need to be addressed . First, in vivo prediction of polyp histology is required to reach an adequate level of accuracy, in order to minimize the possible contribution of postpolypectomy histological examination to the decision - making process . Secondly, a high degree of inter and intraobserver agreement is required, in order to assure its reproducibility . Third, a feasible training and learning curve must be in place, in order to result into an adequate generalizability of the predict - resect - and - discard strategy . Fifth, the appropriateness of the clinical recommendations - i.e., the postpolypectomy surveillance intervals - should not be deteriorated by the exclusion of pathological examination . Sixth, a standardized and formal reporting of in vivo histological prediction must be in place, in order to prevent misuse or eventual medical litigations related with the implementation of this policy . Aim of this review is to address the pros and cons of a predict - resect - and - discard policy based on the data available in the literature . Standard white light endoscopy has been regarded as inaccurate to accurately predict in vivo the histological features of colorectal lesions . Sensitivity has been shown to broadly range between 60% and 90%, with specificity ranging between 40% and 90%.19 - 21 the evolution from standard to high - definition endoscopy disappointingly failed to meaningfully improve the suboptimal white light accuracy in characterizing colorectal polyps.19 the possibility to in vivo differentiate among different histotypes has been initially shown by japanese endoscopists.2,17,18,22,23 briefly, it was exploited the ability of some dyes - such as indigo carmine and methylene blue - to highlight the pit pattern of colorectal lesions at high-(optical) magnification endoscopy.2,17,18,22,23 the pit pattern of both polypoid and nonpolypoid lesions was shown to be intimately associated with the progressive distortion and elongation of colonic glands during colorectal cancer (crc) carcinogenesis or, alternatively, with the evolution towards a serrated - mainly hyperplastic - histotypes.2,17,18,22,23 however, dye - chromoendoscopy failed to be successfully implemented in western countries for several reasons . First, japanese endoscopists appeared to be more prone to implement chromoendoscopy in colonoscopy, because chromoendoscopy had already been routinely adopted for population - based gastric cancer screening.24 secondly, dye spraying has been generally discarded as time - consuming and inconvenient, also when considering the intensive volume of screening colonoscopies performed in western countries.13 thirdly, dye - chromoendoscopy requires a long - lasting learning curve, possibly including several hundreds of examinations.2,16,17,21,22 fourth, chromoendoscopy has been pri - marily exploited for differentiating between endoscopically and surgically resectable submucosal cancer in nonpolypoid lesions rather than for discriminating between nonneoplastic and neoplastic lesions.2,16,17,21,22 fifth, an excessive dye spraying may substantially reduce the visibility of the mucosa surrounding the targeted lesion, potentially affecting the detection rate of more serious lesions . For all these reasons, there was the need of a major breakthrough to expand the success of advanced endoscopic imaging to western countries . As detailed by rastogi et al . In this issue of clinical endoscopy, the advent of electronic chromoendoscopy (ec) - such as narrow band imaging, fuji intelligent color enhancement, and pentax i - scan - opened the door for the in vivo prediction of polyp histology in western countries . First, ec has shown a promising accuracy in differentiating between adenomatous and hyperplastic polyps in several studies (table 1),9,14 - 16,19 - 21,25 - 52 owing to the advantage of being extremely simple and fast to be activated . Secondly, the level of confidence of the operator in the in vivo prediction has been associated with ec accuracy, so that - by restricting the analysis only to the high - confidence diagnosis - ec accuracy may be further improved.9,15,16,47 - 49 third, ec is available on the new generation of endoscopes, so that a widespread implementation of this technique may be expected . Fourth, a very fast and simple training and learning curve has been shown to be feasible, also leading to acceptable rates of inter and intraobserver agreements, thus assuring the generalizability and reproducibility of the in vivo characterization.25,47,53 the efficacy and safety of strategies based on in vivo characterization of colorectal lesions depends on a complex interaction between the accuracy values of ec, the expected prevalence of disease, and the types of surveillance intervals recommended . The main criticism to ec - based prediction is its inability in discriminating between nonadvanced and advanced neoplasia.9,14 - 16,19 - 21,25 - 50,54 since unfavourable histological features - such as high - grade dysplasia or> 20% villous component - predict a higher risk of metachronous advanced neoplasia,55 the identification of unfavourable histological features actually results into the assignment of a more intensive postpolypectomy surveillance (i.e., 3-years vs. -5/10-years).5,6 therefore, ec - based policies should be implemented only when the risk of advanced neoplasia may be marginalized . When comparing the prescreening prevalence of advanced neoplasia - ranging between 4% and 10% - with that of subcentimetric polyps, such prevalence would appear to be marginal - i.e., 1% - in diminutive lesions, whilst ranging between 1% and 5% in 6 to 9 mm lesions.7,8,56 consequently, the actual drawback of a predict - resect - and - discard policy for diminutive lesions would be the lack of characterization of one case of advanced neoplasia every 100 predictions, resulting into a 2/7-year delay in the postpolypectomy surveillance interval . Suggestively, the predict - resect - and - discard policy would still appear safer than the actual nonreferral policy implemented by computed tomography colonography and colon capsule, according to which diminutive lesions, irrespectively of whether (advanced) adenomatous or hyperplastic, should not even sent to post - test polypectomy.57,58 on the other hand, the application of ec - based policies to 6 to 9 mm lesions appears to be more controversial, because of the higher prevalence of advanced neoplasia within these lesions.7,46 however, the much lower prevalence of 6 to 9 mm as compared with 5 mm lesions in the general population would marginalize the impact of these lesions over the efficiency of ec - policies.8 the second criticism to in vivo prediction of polyp histology is the possibility of ec - false - negatives / positives results when discriminating between hyperplastic and adenomatous polyps (table 1).9,14 - 16,19 - 21,25 - 52 this would result in the risk of either anticipating the postpolypectomy surveillance interval in those with an hyperplastic lesion misclassified at ec as adenomatous or delaying it in those with an ec - misclassified adenomatous lesion . The clinical consequences of ec - misclassification would mainly depend on two variables, namely prevalence of adenomatous histotype and type of guidelines adopted . Similarly to advanced neoplasia prevalence, relative prevalence of adenomatous histotype within colorectal lesions is intimately related with polyp size, being about 50% and about 65% in diminutive and small lesions, respectively.7,59,60 based on these values, when assuming a 90% ec - based sensitivity for adenomatous histotype, the negative predictive value for adenomatous histology would equalize a 90% threshold for diminutive lesions, but it would be substantially inferior for 6 to 9 mm polyps . When limiting our analysis to diminutive lesions, polyp prevalence is also likely to be related with polyp location . Because of the well - known prevalence of several tiny hyperplastic lesions in the rectosigmoid tract, the relative prevalence of adenomatous histotype may be expected to be further reduced in this tract, as compared with the remaining colon . In a consecutive series of 235 distal polyps - including 220 5 mm lesions - only 38 were actually adenomatous, corresponding to a 16% overall frequency of the adenomatous histotype.9 because of the inverse relationship between adenomatous prevalence and negative predictive value, the very low disease prevalence in the rectosigmoid tract further reassure on the safety of ec - based policies in this location . The clinical impact of eventual false - positive / negative results at ec would also depend on the type of guidelines adopted . According to the united states and european - based guidelines, patients with one to two tubular subcentimetric adenomas should have their next surveillance examination at 5 to 10 years and 10 years, respectively.5 the impact of this 5 to 10 year difference on the implementation of ec - based policies is dramatic . If a 5-year interval is chosen, any false - positive / negative result at ec would cause an inappropriate anticipated / delayed surveillance interval for hyperplastic and adenomatous lesions, respectively, underlining the necessity for an optimal ec - accuracy in this scenario . On the other hand, if a 10 year interval for patients with one to two tubular subcentimetric adenomas is adopted, any false - positive / negative result would not affect the 10-year surveillance interval, since adenomatous and hyperplastic lesions would share a common 10-year interval recommendation in this scenario . The final criticism to ec - based policies is represented by the impossibility of differentiating between hyperplastic and nonhyperplastic serrated lesions - such as sessile serrated polyps or sessile serrated adenomas lesions - at ec, because of the similar ec characteristics of these lesions.14 however, prevalence of sessile or traditional serrated adenoma has been shown to be very low in subcentimetric lesions, being cumulatively present in 0.3% to 0.5% and 0.8% to 1.3% of diminutive and small lesions, respectively.7,60 moreover, these lesions seem to be associated with a higher risk of synchronous or metachronous advanced neoplasia only when located in the proximal colon or larger than 10 mm.61 based on all these considerations, the american society for gastrointestinal endoscopy (asge) recently developed a preservation and incorporation of valuable endoscopic innovations (pivi) statement for real - time endoscopic assessment of the histology of diminutive colorectal polyps.62 in detail, it was determined that: 1)' for colorectal polyps <5 mm in size to be resected and discarded without pathologic assessment, endoscopic technology (when used with high confidence) used to determine histology of polyps <5 mm in size, when combined with histopathologic assessment of polyps> 5 mm in size, should provide a> 90% agreement in assignment of postpolypectomy surveillance intervals when compared with decisions based on pathology assessment of all identified polyps. '2)' for a technology to be used to guide the decision to leave suspected rectosigmoid hyperplastic polyps <5 mm in size in place (without resection), the technology should provide> 90% negative predictive value (when used with high confidence) for adenomatous histology .' 1)' for colorectal polyps <5 mm in size to be resected and discarded without pathologic assessment, endoscopic technology (when used with high confidence) used to determine histology of polyps <5 mm in size, when combined with histopathologic assessment of polyps> 5 mm in size, should provide a> 90% agreement in assignment of postpolypectomy surveillance intervals when compared with decisions based on pathology assessment of all identified polyps .' 2)' for a technology to be used to guide the decision to leave suspected rectosigmoid hyperplastic polyps <5 mm in size in place (without resection), the technology should provide> 90% negative predictive value (when used with high confidence) for adenomatous histology .' The asge, therefore, recommended two different ec - based policies, namely a predict - resect - and - discard strategy for nonrectosigmoid <5 mm lesions characterized at ec with high - confidence, and a predict - and - do - not - resect policy for rectosigmoid diminutive polyps predicted as hyperplastic at ec with high confidence . 1.62 the ability of ec to meet with the 1 pivi has been tested in a few series (table 2).9,15,16,19,25,46 - 50 when recommending a 5-year interval surveillance for low risk subcentimetric adenomas,5 the majority of the available series failed to match the 1 pivi, especially when ec was performed by less experienced endoscopists . On the other hand, when adopting a 10-year schedule, most of the studies succeeded in meeting the 1 pivi . Intriguingly, when considering a 10 year interval for low - risk subcentimetric adenomas, a different strategy, in theory, would be to do - not - predict - resect - and - discard one to two diminutive lesions without ec - based prediction, reserving ec differentiation only to patients with at least three lesions . However, it is unclear whether the medical community would be ready to resect and discard lesions without having received any prediction about its histology.11 the 2 pivi is rather innovative, since it introduces the predict - and - do - not - resect strategy based on the main assumption that - after ec - prediction of hyperplastic histotype - the possibility of leaving in situ a 5 mm adenoma is too low to warrant the cost of polypectomy itself . Therefore, the predict - and - do - not - resect policy represents a dramatic step forward in rationalizing the exploitation of medical and economic resources at colonoscopy, since it does not only exclude the pathological examination, but also the polypectomy itself from the colonoscopy procedure . When considering the low prevalence of adenomatous component among subcentimetric rectosigmoid lesions, it was not unexpected that the majority of the available studies consistently validated the feasibility of this policy, resulting into> 90% negative predictive values . The exclusion from this strategy of nonrectosigmoid lesions may be also expected to marginalize the risk of leaving high - risk serrated lesions in situ . However, it could also be argued that the drawback of this policy is to accept that 1 in every 10 patients with a rectosigmoid polyp discarded at ec would remain in situ, resulting into the risk of progression of one unresected diminutive ec - misclassified adenoma . At this regard, previous studies addressed the natural history of unresected diminutive or larger lesions in the recto - sigmoid tract, reassuring on the very low risk of progression of these lesions . In detail, hoff et al.63 and hofstad et al.64 followed up 194 diminutive and 253 10 mm polyps detected for 3 and 2 years, respectively . No diminutive polyp reached a> 5 mm size and only 0.5% of 10 mm polyps eclipsed the 10-mm threshold after a 1-year time interval, and no case of severe dysplasia or carcinoma was registered.63,64 in a recent japanese study only 2.9% of 408 subcentimetric lesions followed up for 43.1 months reached a 10 mm size, without the occurrence of any invasive cancer.65 overall these data may be reassuring, when considering the very low risk of discarding an (advanced) adenoma in the rectosigmoid colon . The increasing volume of colonoscopy procedures - related with a growing awareness of the efficacy of crc screening - in a period of economic constraint requires a rational exploitation of the limited medical and financial resources . This is further worsened by the additional volume of surveillance procedure generated by the detection of neoplastic lesions at the screening examination, especially when considering the increase in adenoma detection rate with the evolution of endoscopic technology and the development of quality assurance programs . For this reason, any policy that may simplify and rationalize colonoscopy procedure or work up is useful . Predict - resect - and - discard and predict - and - do - not - resect policies offer the advantage of saving the cost for polypectomy or postpolypectomy pathological examination in a substantial percentage of the cases, as well as to recommend on the same day of the colonoscopy procedure the recommended surveillance interval . In order to meet the desired clinical thresholds, however, ec - based policies require a careful reassessment of the current postpolypectomy surveillance guidelines . When considering the intrinsic limit of ec - accuracy, the recommendation of a 10-year interval for low - risk adenomas would minimize any risk of ec - based false - positive / negative result, prompting for an immediate implementation of these new policies in clinical practice.
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A 64-year - old male with past medical history of hypertension, hyperlipidemia, and peripheral artery disease with left carotid endarterectomy that was done 8 years ago, who was treated for salmonella enteritis 4 months ago with ciprofloxacin 500 mg tablet twice daily for 10 days, presented with complains of nausea, vomiting, and abdominal pain . He had experienced these symptoms on and off since he was treated for enteritis, but worsened prior to admission . On examination, he had a temperature of 103.5f; heart rate, 87 beats / min; blood pressure, 133/85 mm hg; and oxygen saturation 97% on room air . Laboratory workup was normal except for a white cell count of 14,600/mcl and hemoglobin of 11.2 g / dl . (iv) contrast that was done to evaluate for abscess showed a fusiform 3.4-cm infrarenal, abdominal, aortic aneurysm along with an adjacent 2.12.12.7-cm mycotic aneurysm (figs . 1 and 2). After a discussion with vascular surgery team regarding the risk benefits of evar versus open surgery, the patient opted for an interval endoluminal graft placement after completion of antibiotic therapy . After a week of iv antibiotics, repeat ct angiogram of the abdomen was done . He was discharged home with 6 weeks of ceftriaxone and advised to follow - up with infectious disease and vascular surgery for endoluminal graft placement . He successfully underwent interval evar and was doing well until 18 months after evar without any postoperative infectious complications (figs . 3 and 4). The patient had a passion for hunting turtles and reported eating them cooked almost once every 23 months for the past 15 years . Ct angiogram of abdomen and pelvis transverse section showing the mycotic aneurysm . Ct angiogram of abdomen and pelvis sagittal section showing the mycotic aneurysm . Ct angiogram of abdomen and pelvis status post - evar with successful exclusion of the mycotic aneurysm . Ct angiogram of abdomen and pelvis (coronal view) status post - evar with aorto - bi - femoral graft . The salmonellae are a heterogeneous group of bacteria in the genus salmonella of the family enterobacteriaceae . The centers for disease control and prevention (cdc) currently recognizes two species: salmonella enterica (6 subspecies) and salmonella bongori (1 subspecies). Salmonella are usually known to cause gastroenteritis, which rarely requires antibiotic therapy . Immunocompromised and elderly population are prone to develop extra - intestinal complications, the most feared being infected aneurysms of the abdominal aorta (1219). Salmonella typhimurium (serogroup b), salmonella enteritidis (serogroup d), and salmonella choleraesuis (serogroup c) are the common serotypes associated with mycotic abdominal aneurysm (20, 21), s. enteritidis is more common in europe compared to east asia where non - typhoidal salmonella is more common (10, 2226). Salmonella has a predisposition to attach to vascular endothelium, more so in atherosclerotic blood vessels (1). However, the exact mechanism and pathogenesis of salmonella causing the mycotic abdominal aneurysm is still unclear (6). Recurrent and latent infections are possible from salmonella due to mechanisms that promote intracellular survival (27). Mode of transmission of salmonella is fecal oral and vehicle - borne . Infection may result from ingesting water or food that has been contaminated with animal or human feces, or from direct exposure to the same (28). . However, some of the common presenting symptoms are fever, pain, shock, and leukocytosis (6). Widespread use of ct scan to evaluate for unexplained abdominal symptoms and sepsis has led to the early identification of mycotic abdominal aneurysm (6). The food and drug administration (fda) prohibits the selling of turtles with a shell under 4 inches in length in an effort to prevent contact with turtles carrying the salmonella bacteria . The cdc recommends that children, pregnant women, and persons with compromised immune systems avoid contact with reptiles to prevent contact with the salmonella bacteria (29). Small pet turtles are of particular concern because children are more prone to handling them without washing their hands after, and even put the turtles in their mouths (29). Infectious disease specialists estimate that banning small turtles as pets prevents 100,000 salmonella infections in children each year in the united states (29). It is a popular option to treat with at least 6 weeks of oral or parenteral antibiotics (1, 30). However, recent studies have shown that most recurrent infections occurred in the first 612 months after the procedure and the majority of the fatal infections and sepsis - related complications developed after discontinuing the antibiotics (1). These finding are suggestive of a potential benefit from long - term antibiotic therapy for 6 months to 1 year, or lifelong based on a case - by - case basis . Positive blood cultures for salmonella during the initial postoperative period have a favorable outcome compared to non - salmonella - positive blood cultures . The same antibiotics would be less beneficial in patients who have a non - salmonella infection (1, 10, 3133). Open surgery (debridement and surgical resection of the infected aorta and the surrounding tissues, the use of body tissue to cover the infected field, and either an extra - anatomic bypass or in situ interposition graft) followed by long - term antibiotic therapy has been the gold standard treatment but is associated with increased mortality and morbidity (1, 9, 10, 23, 32, 3438). It has a short - term mortality of 2040% although there is not sufficient data to comment on long - term outcomes (10, 23, 3438). Anatomical position of the aneurysm sometimes makes open conventional surgery a less preferred option (1, 9). It has the potential to become a popular alternative to open conventional surgical management, either as a permanent management option, or as a temporary bridge to stabilize the patient prior to open surgery . Insertion of a graft in acute infection prior to completing a course of antibiotics is controversial . Hence, interval evar (after a full course of antibiotic therapy) might help prevent late infection complications . However, in evar, there is a potential nidus for a late infection of the graft and sepsis . A recent retrospective study done by sorelius et al . (1) analyzed that evar is a feasible and durable treatment option for patients with mycotic aneurysms . Post - evar, their study showed a 1-month survival of 91% and a120-month survival of 41%; however, only 19% deaths were due to infection . Non - salmonella - positive blood cultures were associated with increased mortality from late infection (1). In mycotic abdominal aneurysm patients who do not pursue either surgical or endovascular treatment, studies have shown up to a 75% mortality rate from complications related to mycotic aortic aneurysm(6). There is a possibility of selection bias in the study done by huang et al . However, details of the indications leading up to an open surgery versus evar were not mentioned . Based on this case report, it might be appropriate to mention that patients with vascular disease should be discouraged from hunting and handling turtles, for the fear of contracting salmonella and then the risk of mycotic aortic aneurysm . Based on the review of literature, in a patient with salmonellosis having abdominal pain, especially in patients with risk factors such as elderly age, atherosclerosis, and immunosuppressed status, a ct scan of the abdomen should be done to detect a possible mycotic aneurysm (12). Ct is excellent in preoperative and postoperative evaluation of aneurysms and their potential complications (39). Clinical significance: have a low threshold to get a ct scan of the abdomen in salmonellosis patients with risk factors and abdominal symptoms.in salmonella mycotic abdominal aneurysm, there could be potential benefit from long - term antibiotic therapy for 6 months to 1 year or lifelong . Evar has the potential to become a popular alternative to open conventional surgical management of mycotic abdominal aneurysm as late infections are a major complication of the latter . Have a low threshold to get a ct scan of the abdomen in salmonellosis patients with risk factors and abdominal symptoms . In salmonella mycotic abdominal aneurysm, there could be potential benefit from long - term antibiotic therapy for 6 months to 1 year or lifelong . Evar has the potential to become a popular alternative to open conventional surgical management of mycotic abdominal aneurysm as late infections are a major complication of the latter.
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Vaccine programs are underpinned by a rigorous science determining their efficacy and safety in populations . There needs to be a similar level of commitment to identifying and testing the interventions designed to increase uptake of vaccines among vaccine - hesitant parents . Accordingly, governments and research agencies need a greater investment in the strategic direction, capacity building, research and evaluation to meaningfully address vaccine hesitancy . Vaccine hesitancy involves developing a deep understanding of the psychological and social dimensions of vaccine acceptance, building good measures that can identify and monitor patterns of vaccine hesitancy in populations and over time, and systematically testing interventions using valid and reliable outcome measures . Interventions should consider communication interventions operating at the individual, family, and community level . They should be economically viable and shown to not cause harm prior to their implementation . Only good monitoring of vaccine acceptance attitudes will determine true trends in the prevalence of vaccine hesitancy . Many commentators consider increasing rates of disease, vaccine exemptions, and alternative vaccine schedules or reductions in coverage as an indication of declining vaccine acceptance . While these epidemiological and behavioral outcomes may be indicative, they are influenced by a range of possible factors . With respect to changes in coverage, parsing acceptance from access is essential . This most basic differentiation is hampered by the lack of national monitoring of both acceptance attitudes and perceptions of financial, physical, and social cultural barriers to access . Few countries undertake active monitoring of actual vaccine acceptance over time using valid and reliable measures deployed in populations large enough to enable confident conclusions . These returns were most clearly demonstrated with the uk government's yearly monitoring of mothers attitudes toward vaccination between 1991 and 2012 enabling program managers to anticipate shifts in public confidence following the mmr - autism scare . Two major influences on vaccine acceptance and rejection emerge from the literature to date: social norms and provider interactions . With regard to the former, rejection of vaccination tends to cluster in communities typically characterized by alternative or religious belief systems . To the extent that rejection and hesitancy toward vaccination is a community phenomenon, solutions need to be considered at the community level . Community - based interventions are already utilized in low- and middle - income countries, where they may be used to build trust and increase community participation in populations that are difficult to reach with mass media or standard health services . Adversarial and/or top - down approaches can polarize communities and alienate parents less favorable toward vaccination . In contrast, communication that engages communities in dialog through local opinion leaders or peer groups has the potential to build community support and advocacy for the benefits of vaccination . A second major influence on whether a hesitant parent accepts or rejects a vaccine is the provider interaction . A number of approaches have been advocated yet, as noted by williams, the evidence base for effectiveness is wanting . Confident recommendation combined with respectful engagement, narrative and personalized approaches that address the needs of vaccine hesitant parents appears to be the most constructive way . Reviews should also appropriately assess the quality of included studies to ensure that the evidence base is not founded on low - quality studies . The outcomes measured need attention . As williams notes, outcome measurement across studies is inconsistent, making it difficult to build the evidence base through systematic reviews of effects . We need a comprehensive understanding of both process outcomes (e.g., knowledge or intent to vaccinate) as well as endpoints (e.g., timely and complete vaccination) in order to determine how and at what stages specific communication interventions affect vaccination attitudes and behavior . Measuring process outcomes allows the identification of components of the package that are effective and indicates whether the end effect is related to implementation issues or to the intervention itself . Along with vaccination timeliness and completion, it is prudent to measure outcomes of communication such as informed decision - making, satisfaction, and decisional conflict . These reflect good and ethical process and are essential if we are to ensure sustainable trust at an individual and population level . A committed, confident and competent vaccination workforce is integral to ensuring high vaccine coverage . . There should be sufficient time devoted to vaccination in university curriculum and continuing education . If health professionals have a nuanced understanding of vaccines and vaccine hesitancy, they will be better prepared for a guiding partnership in vaccine decisions with parents . Another emerging priority is better engagement of maternity care nurses and complementary and alternative medicine practitioners who have a voice at crucial times of vaccine decision making in key groups . For over two hundred years, industrialized countries have sustained the political will, financial support, purchasing structures, cold chain, program management, workforce capacity, and communications to ensure that most children get most or all the vaccines recommended to them . Vaccine hesitancy presents particular challenges because the attitudes and beliefs underlying it may be self - sustaining and not be amenable to centralized and administrative efforts . Undoubtedly though, the global vaccination community has the capacity to address vaccine hesitancy . It will be able to do this if there is sufficient political will, professional commitment, and research investment to develop and evaluate new and innovative solutions that make a meaningful difference.
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Hepatocellular carcinoma (hcc) is a common malignancy and is now one of the major causes of death in asian countries . Generally, hccs are often asymptomatic, but fatigue, abdominal distention and low - grade fever however, fever as a primary symptom in hcc is relatively rare . Additionally, high crp in laboratory results is also rare . Hcc with pyrexia has been reported only in a few studies [24] and the cause of pyrexia has remained unknown . Recently, increased production of proinflammatory cytokines and chemokines, such as interleukin-1 (il-1), interleukin-1 (il-1), interleukin-6(il-6), interleukin-8(il-8), and tumor necrosis factor- (tnf-) have been reported to be febriferous in acute and chronic inflammatory disease [5, 6]. These cytokines are produced by immune cells including various populations of lymphocytes macrophages and other cells . Among the proinflammatory cytokines, il-8 is a neutrophil chemotactic factor and involved in tumor proliferation and migration, angiogenesis in malignant tumors . This is often accompanied by rapid growth and poor prognosis in malignant tumors [713]. We herein discuss a case of hcc accompanied by prolonged spiking fevers, which disappeared after tumor resection . A 50-year - old man with a history of liver dysfunction was admitted to iizuka hospital with the chief complaint of a prolonged fever and general fatigue in october, 2005 . Hematological laboratory data on admission were as follows: wbc 9670/ l, alp 552 u / l, crp 16.7 mg / dl, and afp 11.1 ng / ml . Hbs - antigen was positive (table 1). A computed tomography (ct) showed a peripherally enhanced low density mass 7.5 cm in diameter, which located in segment 6 in the right lobe of the liver (figure 1(a)). The tumor was accompanied with tumor thrombus to the posterior branch of portal vein (figure 1(b)). Celiac angiography showed this lesion was hypervascular (figure 1(c)) and portal vein tumor thrombus in posterior segment was observed in the portal phase (figure 1(d)). Patients have chronic hepatitis b and then tumor was hypervascular and accompanied with portal thrombus, which is the feature of hcc, therefore a preoperative diagnosis was made as hcc with portal vein thrombus and nonsteroidal anti - inflammatory drugs were used to reduce the fever . A single nodule of disseminated cancer cells was found near the posterior segment on the abdominal wall during the operation . Posterior segmentectomy of the liver and thrombectomy including the dissemination was performed . On the cut section, the noncancerous liver tissue showed a moderate chronic inflammatory infiltrate in the fibrous stroma, diagnosed as liver fibrosis . Immunohistochemical stain of il-8 was performed with paraffin embedded sections of liver tissue using mouse monoclonal antibody at dilutions of 1: 100 (assay designs, ann arbor, usa). The subsequent reaction was performed by the peroxidase labeled streptavidin - biotin technique using histofine sab - po kit (nichirei, tokyo, japan). Il-8 was observed in the cytoplasm of cancer cells (figure 3(b)). After the tumor resection, fever disappeared and crp dropped gradually from 16.7 to 2.7, but the patient had recurrence with multiple liver metastasis and pleuritis carcinomatosa and died on february 2, 2006, 79 days after the operation . In 1991, okuda et al . Reported five patients with hcc and pyrexia, for an incidence of less than 1%, in japan . He also mentioned that very poorly differentiated sarcomatoid hcc may frequently appear with pyrexia and leukocytosis mimicking liver abscess . In 1995, hayashi et al . Reported hcc with pyrexia laboratory data revealed high crp . Nevertheless, in both case reports, the mechanism of a fever was not revealed . In our case, the sarcomatoid change of the cancer cell was not detected, only poorly differentiated hcc . It is well known that cancer cells produce humoral factors and cause the paraneoplastic syndrome . Among the clinical symptoms and laboratory data, fever and high crp, which is not commonly observed in the patients with hcc, is suspected to be due to humoral factors, especially inflammatory cytokine . Il-8 is a proinflammatory cytokine whose principal role in infection and inflammation appears to be the recruitment and activation of circulating and tissue neutrophils to the site of tissue damage . It has been demonstrated that il-8 is produced by a wide variety of cell types in vitro, including endothelium, monocytes, eosinophils, astrocytes and keratinocytes . In sepsis patients, for example, il-8 concentrations have been reported as being markedly elevated at diagnosis and remaining high during the course of the illness . This symptom is induced by many humoral factors, including il1, il-6, il8 and tnf. Zampronio ar reported that il-8 induced fever by prostaglandin independent mechanism . Il-8 resulted in a dose - dependent increase of crp, a decrease in the production of transferring and prealbumin . Therefore, il-8 is related to pyrexia in parts, not all . In other cancer, we examined by immunohistochemical staining of il-8 and tumor tissue stained positive for il-8 in this case . Akiba et al . Reported that a high il-8 level in hcc had a significantly higher frequency of portal vein invasion and venous invasion and bile duct invasion . In vitro reported il-8 production was enhanced progressively with escalating severity of hepatitis and the development of hcc and the level of il-8 were significantly increased in patients with advanced hcc with distant metastasis, and then this leads to poor prognosis . Ren et al . Reported serum il-8 level was correlated with tumor size and tumor stage, and then il-8 level was a significant prognostic factor in terms of disease - free survival and overall survival . Additionally, elevated il-8 levels in the drainage vein of colorectal cancer are related to the occurrence of hepatic metastasis . This case presented portal vein tumor thrombus, bile duct invasion, intrahepatic metastasis, peritoneal dissemination, early tumor recurrence, rapid tumor regrowth and finally a poor prognosis . In fact, all patients with il-8 production do not have a high fever, the serum concentration of il-8 or the degree of neutrophil infiltration will be concerned with pyrexia . In many cancers such as esophageal cancer, gastric cancer and colorectal cancer, serum crp was known as a prognostic indicator [2023]. In particular, some reported that serum crp levels indicated a poor prognosis in hcc patients and portal vein invasion was significantly higher in the serum crp - positive group [24, 25]. According to the in vitro previous studies, cultured hcc can produce crp that is regulated in part by proinflammatory cytokines, such as il-6, il-8 and tnf- [18, 26, 27]. Among the many proinflammatory cytokines, recent report il-6 mediates cell cycle arrest in hepatocellular carcinoma through a stat 3-dependent pathway hcc produce proinflammatory cytokines such as il-8 by autocrine or paracrine, which lead to crp production and inflammatory cascade or tumor progression . In this report we discussed a patient who had poorly differentiated hcc with pyrexia and high crp . Il-8 production was histologically revealed in this case and may have contributed to the high fever, high inflammatory reaction and poor prognosis.
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The term fibromatosis refers to a group of benign soft tissue tumors (fibromas), which have certain characteristics in common, including absence of cytologic and clinical features of malignancy, a histology consistent with proliferation of well - differentiated fibroblasts, an infiltrative growth pattern, and aggressive clinical behavior with frequent local recurrence . Herein we report this unusual presentation of benign fibromatosis over the dorsum of both feet . A 40-year - old female patient presented with multiple raised growths over both feet since four years . Contractures, bony deformities and keloids were absent . On examination, symmetrical, well - defined, firm nodules coalescing to form lobulated plaques were noted over the dorsum of both feet extending from base of the 2, 3, 4, and 5 toes to the mid - foot [figures 1 and 2]. Radiographs of both feet showed no bony deformity and ultrasound of both feet showed no significant soft tissue thickening . An elliptical biopsy of lesion taken for histopathology showed epidermis with hyperkeratosis, acanthosis, mild parakeratosis and prominent granular layer . Superficial dermis was unremarkable and deep dermis showed fibroblastic proliferation with variable vascularity and lack of inflammatory infiltrate, and thick bundles of irregular collagen characteristic of fibromatosis [figures 3 and 4]. With these clinical and histopathological features, the patient was advised topical keratolytics along with intralesional steroids for 2 months with minimal response . The patient was counseled about the benign nature of the disease and measures for lifestyle modification and proper footwear . Multiple well - defined firm nodules coalescing to form lobulated plaques distributed in a symmetrical pattern over dorsum of both feet, extending from base of the 2, 3, 4, and 5 toes to the the mid - foot close - up view of the lesion showing diffuse lobulated plaque over the dorsum of left foot epidermis showing hyperkeratosis and acanthosis with deep dermis showing fibroblastic proliferation with thick bundles of irregular collagen (hematoxylin and eosin 4) epidermis showing hyperkeratosis, acanthosis, mild parakeratosis, and a prominent granular layer . Superficial dermis was unremarkable and deep dermis showed fibroblastic proliferation with variable vascularity and lack of an inflammatory infiltrate, with thick bundles of irregular collagen (hematoxylin and eosin 40) the musculoskeletal fibromatosis represent a wide range of fibroblastic to myofibroblastic proliferations that are grouped together because of their similar pathologic appearances . The clinical behavior of these tumors is intermediate between that of benign and malignant fibrous lesions; they commonly manifest infiltrative growth, resulting in frequent local recurrence, but lacking metastatic potential . The world health organization committee for classification of soft tissue tumors in 2002 categorized these lesions as superficial or deep based on their anatomic location . Superficial fibromatosis (palmar, plantar, penile, and knuckle pads) and deep fibromatosis (nonmetastasizing fibrosarcoma, and desmoids tumor), which are rapidly growing tumors involving the muscular structures or aponeuroses . Clinically, it may present as solitary or multiple lesions affecting one or many sites; the prevalence of palmoplantar involvement may vary 0 - 28 percent of cases . The superficial fibromatosis, whether they occur in adults (palmar and plantar) or children (calcifying aponeurotic fibroma, lipofibromatosis, and inclusion body fibromatosis), are typically small lesions that grow slowly . The deep fibromatosis in both adults (desmoid type and abdominal wall) and children (fibromatosis colli and myofibroma and myofibromatosis) are usually larger and often enlarge more rapidly . The diagnosis of deep fibromatosis may be suggested by their clinical characteristics, particularly anatomic location and patient age: fibromatosis colli that typically involves the lower neck and sternocleidomastoid muscle in a young child; abdominal wall fibromatosis that manifests as a mass involving the rectus abdominus muscle and is often related to pregnancy; or myofibromatosis that occurs as multicentric disease in a young child; palmar fibromatosis, a fibromatous hyperplasia of the palmar aponeurosis characterized by nodular thickening of the fascia associated with flexion contractures of one or more digits; plantar fibromatosis comprising single or multiple painful nodules over the medial half of mid - foot with a tendency to ulcerate that may be locally invasive and can recur; penile fibromatosis, characterized by one or more dense fibrous plaques on the penile shaft that occur as an isolated abnormality or as part of polyfibromatosis; knuckle pads, characterized by circumscribed thickening overlying the proximal interphalangeal joint; desmoid tumors - histologically benign but locally aggressive fibrous neoplasms originating from the musculoaponeurotic structures with often nonspecific findings . Diagnosis relies on histopathologic findings characterised by multinodular cellular proliferations of uniform, plump, spindle shaped fibroblastic cells within a collagenous stroma . Longstanding lesions are less cellular and contain increased amounts of dense collagen, suggestive of fibromatosis . Cross - sectional imaging (ultrasonography, computed tomography, or magnetic resonance imaging [mri]) reveals lesion location, extent, and involvement of adjacent structures and thus is useful for tumor staging and evaluation of local recurrences . Additional mri features that are related to underlying pathologic characteristics provide increased specificity for the diagnosis of musculoskeletal fibromatosis . Treatment of musculoskeletal fibromatosis may range from conservative management to surgical resection and is influenced by the specific diagnosis and extent of the lesion determined at imaging evaluation . Local recurrence is common after surgical resection, owing to the infiltrative growth seen pathologically in these lesions . Wide excision of fascia is the treatment of choice for solitary painful lesions or lesions causing flexion contractures . Recurrences are common in aggressive fibromatosis, but not so in superficial benign fibromatosis . In a case series of 14 patients with plantar fibromatosis, it was noted that symptoms improve with time with conservative management and intralesional corticosteroids may not always cause subsidence of symptoms . Among the nonsurgical treatments, radiation therapy, vitamin e, local injection of calcium channel blockers, interferon, corticosteroids or collagenase, and colchicine, we used intralesional injection of triamcinolone in escalating dose of 10 and 40 mg / ml every three weeks along with topical salicylic acid 12% and urea for local application twice daily that showed minimal response . This case report presents the benign nature of the condition, its relative resistance to treatment and an uncommon symmetrical presentation of fibromatosis over the dorsum of both feet.
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Brucellosis is a frequently encountered zoonotic disease in the developing countries, and considered as an important public health problem . People can be contracted especially through contaminated meat, milk, and dairy products as well as direct contact with the excrements or body secretions of the infected animals . Are small, gram negative, facultative, intracellular, and pathogenic bacteria; they invade mononuclear phagocytic system cells and proliferate there . Brucellosis is an inflammatory disease that can infect any organs or systems in the body . It is diagnosed through the clinical, serological, and microbiological test results of the patients (1). The acute phase reactants increase as a result of the inflammatory process in brucellosis (1, 2). In endemic regions; however, the laboratory conditions may not always be suitable or sufficient for diagnosis . Since the second antibody titer, assessed in the follow - up after treatment, may remain at a high level, it is difficult to decide whether to stop or continue the medical treatment (1). Therefore, authors believe that additional diagnosis methods may be useful in the follow - up process . Leukocyte count and high sensitive c - reactive protein (hs - crp) are frequently used as inflammatory markers in patients diagnosed with brucellosis . Besides these markers, the current study investigated whether the values of mean platelet volume (mpv) and red blood cell distribution (rdw) could be considered as surrogate markers during the illness phase . Mean platelet volume (mpv) is a measure of platelet size . As part of the routine complete blood count test cycle, mpv is generated by full blood count analyzers but mostly overlooked by clinicians (3). Mpv is one of the most commonly used surrogate markers of platelet function . Comparing with small platelets, large ones contain more granules, aggregate with collagen more quickly, have higher thromboxane a2 levels, and express more glycoprotein ib and iib / iiia receptors (4 - 6). It reveals the presence of inflammatory burden and disease activity in many diseases including preeclampsia, acute pancreatitis, unstable angina, myocardial infarction, and cases of systemic inflammation such as ulcerative colitis and crohn s disease (7). As for rdw, it is a measure of heterogeneity in the size of circulating red blood cells . It is one of the standard complete blood count components, and is calculated as a percentage by dividing the standard deviation of the red cell volume by the mean corpuscular volume . Various studies revealed the clinical implications of rdw about the presence of various pathologies such as inflammatory bowel disease, celiac disease, pulmonary embolism, and coronary artery disease . Moreover, inflammatory and infectious pathological diseases such as acute pancreatitis, bacteremia, sepsis, and septic shock are proven as predictive values (8). In a study by lippi et al . (9), a graded association of rdw with hs - crp and erythrocyte sedimentation rate were reported, independent of various confounding factors . Besides inflammation, oxidative stress although erythrocytes possess a great antioxidant capacity and regularly serve as the chief oxidative sink, they are liable to oxidative damage which reduces cell survival (10). According to a population - based study (11), higher rdw were independently associated with poorer pulmonary function . Nevertheless, it still unknown whether rdw is a simple marker and not a mediator of carotid artery atherosclerosis . Indeed the discovery of a putative causative mechanism is prevented by the lack of epidemiological studies which would reveal the presence of an association between atherosclerosis and anisocytosis . Among the studies published to date, we encountered a single prospective study that examined the mpv, rdw and hs - crp values of patients with acute brucellosis and those convalescing from brucellosis . The current study aimed to investigate the mpv, rdw, and crp values as inflammatory markers of the disease (8). The data of 250 patients diagnosed with in the university hospital, together with the data of 101 healthy controls were assessed . The study was approved by the local ethics committee of baskent university, and in conformity with the helsinki declaration, all patients signed the informed consents (ka08/198). The study evaluated 88 patients with the history of acute brucellosis who were in their first first - year follow - up . The study included patients over the age of 18 diagnosed with brucellosis and followed - up from 2008 to 2014 . In the clinic, the patients diagnosed with brucellosis had high fever, chills, shivering, fatigue, sweating, and muscle and joint aches . The microbiological and serological tests were performed on subjects blood samples . Those with an agglutination titer 1/160 and/or brucella spp . The patients participating in the study were treated with doxycycline (six weeks), rifampicin (six weeks), or streptomycin (21 days). The crp level of the subjects was normal, and they did not have any complaints . The leukocyte counts and the mpv, rdw and high sensitive c - reactive protein (hs - crp) values of the patients were compared with those of the controls . The study excluded patients with abnormal renal and liver function test results or with other infectious diseases and inflammatory conditions . Blood samples were analyzed in the hematological laboratory of the hospital, using standard tubes containing 2 ml blood and 0.04 ml of 7.5% k3 salt of ethylene di amine tetra acetic acid (edta). Since the mpv values could have increased under the influence of edta, the measurements were performed within two hours (12). Afterwards, the serum samples were separated from the cells by placing them in the centrifuge for ten minutes at 3000 rpm . The patients were diagnosed with brucellosis through the standard tube agglutination test (standart tube agglutination, spinreact, spain), the coombs test (1/160) and/or brucella spp . An electronic cell counter (cell - dyne 3700, abbott, abbott park, il, usa) was used to determine the wbc counts . Were used to analyze the hematological parameters, which included hemoglobin (hb, range 14 - 18 g / dl for male, 12 - 16 g / l for female), white blood cell count (wbc, range 4.5 - 11 109/l), platelet count (plt, range 150 - 400 109/l), mpv (range 7 - 12 fl) and rdw (range 11.6 - 15.5%). Spectrophotometric methods (abbott aeroset, tokyo, japan) were used tomeasure serum crp levels . The spss software was employed to carry out the statistical analyses . Comparing the groups, the t -test and chi - square tests were used for continuous categorical variables, respectively . Mann - whitney u test was used to compare nonhomogeneous groups in pairs . In order to observe the correlation between mpv and the other variables, a simple correlation test (the numeric values were expressed as mean sd, and the level of significance as p <0.05 . Blood samples were analyzed in the hematological laboratory of the hospital, using standard tubes containing 2 ml blood and 0.04 ml of 7.5% k3 salt of ethylene di amine tetra acetic acid (edta). Since the mpv values could have increased under the influence of edta, the measurements were performed within two hours (12). Afterwards, the serum samples were separated from the cells by placing them in the centrifuge for ten minutes at 3000 rpm . The patients were diagnosed with brucellosis through the standard tube agglutination test (standart tube agglutination, spinreact, spain), the coombs test (1/160) and/or brucella spp . Culture growth . An electronic cell counter (cell - dyne 3700, abbott, abbott park, il, usa) was used to determine the wbc counts . Were used to analyze the hematological parameters, which included hemoglobin (hb, range 14 - 18 g / dl for male, 12 - 16 g / l for female), white blood cell count (wbc, range 4.5 - 11 109/l), platelet count (plt, range 150 - 400 109/l), mpv (range 7 - 12 fl) and rdw (range 11.6 - 15.5%). Spectrophotometric methods (abbott aeroset, tokyo, japan) were used tomeasure serum crp levels . The spss software was employed to carry out the statistical analyses . Comparing the groups, the t -test and chi - square tests were used for continuous categorical variables, respectively . Mann - whitney u test was used to compare nonhomogeneous groups in pairs . In order to observe the correlation between mpv and the other variables, a simple correlation test (the numeric values were expressed as mean sd, and the level of significance as p <0.05 . The study included 351 subjects, which 250 of them were categorized in the acute brucellosis group, and 101 in the control group . Among the 250 patients with brucellosis, 115 were male and 135 were female, while 50 of the 101 controls were male and 51 were female . The blood values of the 88 patients were recorded for further follow - ups a year after treatment . There was no significant difference between the acute brucellosis and control groups regarding age or gender (p> 0.05) (table 1). The mean leukocyte count was 7.3 2.9 and 7.4 2.0 (10/mm) in the ab and the control groups, respectively . The leukocyte count was insignificantly higher than that of in the control group (p> 0.05). The mean leukocyte count found in the first year follow - up of the patients with brucellosis was 7.1 2.0 (10/mm). When the leukocyte counts in the acute phase and in the first - year follow - up were compared, no difference was observed and the counts were similar to those of the control group (p> 0.05). The mean crp levels were 32.57 53.20 mg / dl, and 4.81 4.89 mg / dl in the ab and control groups, respectively . The crp level in the ab group was significantly higher compared with that of the control group (p <0.05). In the follow - up year, the crp values remained in the normal range: 5.44 8.91 mg / dl (p> 0.05). The mean mpv levels were 7.64 1.30 fl, and 7.67 1.29 fl in the ab and control groups, respectively . Similar to the control group, the mpv level remained within the normal range in the ab group; this result was statistically significant (p> 0.05). In the follow - up year the mpv value was 7.65 1.54 fl, same as those of the acute phase and the control group (p> 0.05). The mean rdw levels were 16.24 2.14% and 15.90 1.45% in the ab and control groups, respectively . The rdw level was not significantly higher in the ab group compared to that of the controls . In the follow - up year, there was insignificant difference between the rdw value in the ab and control groups (16.10 1.79%) (p> 0.05) (tables 1 and 2). While the crp values of the patients in the acute phase were higher compared to those of the controls, their mpv and rdw values had no differences . Similarly, the leukocyte counts of the brucellosis patients in the acute phase and during the follow - up did not differ, and were not different from those of the control group . When the blood parameters were compared after a year of follow - up, it was observed that the mpv, rdw and leukocyte values had not changed, while the crp values had improved as expected in the period of convalescence . It was found that the leukocyte count in acute brucellosis patients correlated with mpv and rdw (r = -0.149, p = 0.009; r = + 0.177, p = 0.002; r = + 0.128, p = 0.015, respectively). It was also observed that the age parameter correlated with crp (r = + 0.272, p <0.001, respectively), and the leukocyte count correlated with crp and rdw (r = + 0.438, p <0.001; r = -0.124, p = 0.029, respectively) (table 3). Although the differences in some of these parameters appeared to be statistically significant in the former studies because of negative factors like insufficient size of the patient group, examining 250 patients with brucellosis in the recent decade during the acute phase and in the first - year follow - up showed that the mpv and rdw values of the patients failed to yield significant results in diagnosis and treatment . Abbreviations: crp: c - reactive protein, mpv: mean platelet volume, rdw: red blood cell distribution . Data are presented as mean sd . Abbreviations: crp: c - reactive protein, mpv: mean platelet volume, rdw: red blood cell distribution . Data are presented as mean sd . Abbreviations: crp: c - reactive protein, mpv: mean platelet volume, rdw: red blood cell distribution . The mean leukocyte count was 7.3 2.9 and 7.4 2.0 (10/mm) in the ab and the control groups, respectively . The leukocyte count was insignificantly higher than that of in the control group (p> 0.05). The mean leukocyte count found in the first year follow - up of the patients with brucellosis was 7.1 2.0 (10/mm). When the leukocyte counts in the acute phase and in the first - year follow - up were compared, no difference was observed and the counts were similar to those of the control group (p> 0.05). The mean crp levels were 32.57 53.20 mg / dl, and 4.81 4.89 mg / dl in the ab and control groups, respectively . The crp level in the ab group was significantly higher compared with that of the control group (p <0.05). In the follow - up year, the crp values remained in the normal range: 5.44 8.91 mg / dl (p> 0.05). The mean mpv levels were 7.64 1.30 fl, and 7.67 1.29 fl in the ab and control groups, respectively . Similar to the control group, the mpv level remained within the normal range in the ab group; this result was statistically significant (p> 0.05). In the follow - up year the mpv value was 7.65 1.54 fl, same as those of the acute phase and the control group (p> 0.05). The mean rdw levels were 16.24 2.14% and 15.90 1.45% in the ab and control groups, respectively . The rdw level was not significantly higher in the ab group compared to that of the controls . In the follow - up year, there was insignificant difference between the rdw value in the ab and control groups (16.10 1.79%) (p> 0.05) (tables 1 and 2). While the crp values of the patients in the acute phase were higher compared to those of the controls, their mpv and rdw values had no differences . Similarly, the leukocyte counts of the brucellosis patients in the acute phase and during the follow - up did not differ, and were not different from those of the control group . When the blood parameters were compared after a year of follow - up, it was observed that the mpv, rdw and leukocyte values had not changed, while the crp values had improved as expected in the period of convalescence . It was found that the leukocyte count in acute brucellosis patients correlated with mpv and rdw (r = -0.149, p = 0.009; r = + 0.177, p = 0.002; r = + 0.128, p = 0.015, respectively). It was also observed that the age parameter correlated with crp (r = + 0.272, p <0.001, respectively), and the leukocyte count correlated with crp and rdw (r = + 0.438, p <0.001; r = -0.124, p = 0.029, respectively) (table 3). Although the differences in some of these parameters appeared to be statistically significant in the former studies because of negative factors like insufficient size of the patient group, examining 250 patients with brucellosis in the recent decade during the acute phase and in the first - year follow - up showed that the mpv and rdw values of the patients failed to yield significant results in diagnosis and treatment . Abbreviations: crp: c - reactive protein, mpv: mean platelet volume, rdw: red blood cell distribution . Data are presented as mean sd . Abbreviations: crp: c - reactive protein, mpv: mean platelet volume, rdw: red blood cell distribution . Abbreviations: crp: c - reactive protein, mpv: mean platelet volume, rdw: red blood cell distribution . Brucellosis is a zoonotic systemic inflammatory disease, which is particularly encountered around the mediterranean (13). The role of platelets in the pathophysiology of brucellosis is not demonstrated yet . In this context, the current study mainly aimed to compare the mpv levels in the acute and post - treatment phases of brucellosis with those of the control group . The present study showed that the mpv and leukocyte values of patients with acute brucellosis were not different from those of the controls . Infections, and particularly respiratory, urinary, gastrointestinal, bone and meningeal infections, affect the thrombocyte count and functions in various ways . While mild anemia and leukopenia are frequently observed in brucellosis, isolated thrombocytopenia and pancytopenia are found less often . There is usually an association between these complications and acute infections (8, 14). Several reports indicate that elevated wbc count is generally the earliest laboratory result to reveal the presence of inflammation and leukocytosis (15, 16). The current study indicated that the leukocyte count of patients with acute brucellosis remained within the normal range without early inflammatory marker . Crp is a sensitive acute - phase protein; and increased in all acute inflammatory processes, however it lacks specificity . It was observed that the leukocyte count in patients with acute brucellosis did not differ from those of the control group, while the crp level was higher (table 1). Thus, it proved to be a good marker to diagnose and monitor the efficiency of the treatment (17 - 19). The fact that the crp values of the patients with acute brucellosis in the current study were high showed that it was still a very valuable inflammatory marker . The role of platelets in brucellosis pathophysiology is not demonstrated yet . In this context, the current study mainly aimed to compare the mpv and rdw levels in the acute and post - treatment phases of brucellosis with their levels in the control group . Moreover, there was a correlation between mpv and the degree of platelet activation and inflammatory responses . Platelet distribution width (pdw) is a direct measurement of the platelet size variability . The most frequently used measure of platelet size, mpv, is a simple marker of platelet function and activation (20). Mpv is accepted as a suitable indicator of platelet activation (21, 22). Although activated platelets normally release antibacterial peptides (23), there is evidence suggesting the presence of certain pathogens that can exploit activated platelets by binding to their surfaces to start or spread an infection (24). Moreover, previous studies reported that mpv changes were associated with various non - infectious inflammatory processes, a condition implying that such changes may indicate disease activity in the inflammation (25 - 27). However, the current study found no difference between the patients with acute brucellosis and the controls regarding the leukocyte, mpv, and rdw values; a result implying that these parameters are not significant markers to diagnose and treat this disease (table 1). Compared with other inflammation markers, the overall accuracy of mpv to predict diseases was generally found superior in the literature . There was a correlation between mpv and crp . High mpv levels also occur in infectious diseases like pulmonary tuberculosis, cchf, and hydatid cyst disease (30 - 32). On the other hand, lower mpv levels were detected by some researchers in active inflammatory bowel disease, rheumatic arthritis, ankylosing spondylitis, acute pancreatitis, and appendicitis (7, 8, 25, 33). What all these conflicting results indicate is that both higher and lower mpv levels may be of diagnostic and prognostic value for various inflammatory diseases . To the authors' best knowledge, the current study was the first to report decreased mpv level in patients with acute brucellosis comparing the control group . As indicated above, examined the mpv values of 39 patients and found the values of 7.84 1.15 fl in the acute phase and 7.83 0.9 fl in the post - treatment phase (1). While these values were within the normal range of mpv, the researchers detected a significant difference comparing them with the mpv values of the controls . However, when the current study examined the mpv values found in the prospective study on the 250 patients with brucellosis followed up in the last decade, no significant differences were observed among the controls, and the values were within the normal range . In another study by kucukbayrak et al, the main mpv value of 40 patients with brucellosis was 7.58 1.96 fl in the beginning of the treatment and 7.90 1.96 fl at the end . They showed that all the values remained within the normal range, but the difference between the pre- and post - treatment values was statistically significant (14). The current study results on mpv were in disagreement with the results of these two studies . Although most studies in the literature reported mpv as an inflammatory marker in many infectious and rheumatological diseases as well as a guiding parameter in diagnosis and treatment (7, 8, 19, 25 - 27, 30, 32, 33), the present study determined that it is not a significant inflammatory marker for brucellosis patients . A study by lippi et al . (9) reported a graded association between rdw and high - sensitivity c - reactive protein and erythrocyte sedimentation rate, independent of various confounding factors . Besides inflammation, oxidative stress erythrocytes have immense antioxidant capacity and serve as the chief oxidative sink, but they are vulnerable against oxidative damage which decreases cell survival (34). Although kucukbayrak et al . Reported high rdw levels in patients with brucellosis before treatment and following it (8), the current study with 250 patients found that the rdw values remained within the normal range before and after treatment, without any statistically significant difference in comparison to those of the control group . Thus, the current study showed that rdw is not a reliable marker in the diagnosis and treatment of patients with brucellosis . The current study results suggested that these values do not play an important role in the diagnosis and treatment of brucellosis . Among the studies in the literature on the inflammatory markers of mpv, rdw, leukocyte count and crp for brucellosis, the present study has the greatest number of patients, and stands as the only prospective study made so far . It was found that mpv and rdw values are not surrogate markers in the diagnosis and treatment of brucellosis . Among other inflammatory markers, high crp is still the most valuable marker for the treatment and follow - up of brucellosis . In contrast, leukocyte count was not found a significant inflammatory marker during the acute phase . The data in the literature regarding this subject generally remain controversial, since they were obtained from patient groups of limited size . The current study was distinguished by the fact that it was performed on the largest patient group so far . It may still be possible for mpv and rdw to act as guiding parameters to diagnose brucellosis . There is still a need for further prospective, multicenter studies with a large sample size to fully clarify the issue.
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Initially uncovered by experiments aimed at understanding ifn and ifn- induced transcriptional activation, a new pathway of signal transduction from the cell surface to genes in the nucleus has been recognized in last decades of 19th century . The pathway is called the jak - stat pathway . Similar to other pathways, association of ligands to their receptors leads to activation of one of the jak family of tyrosine kinases associated with a trans - membrane receptor, and subsequently leads to the phosphorylation on tyrosine of one or more of a family of latent cytoplasmic transcription factors called stats . These latter proteins perform a dual role, first as signal transducers by acting as substrates of the jaks, and after phosphorylation and nuclear translocation, by acting as transcriptional activators . While ifn and ifn were the first polypeptide ligands described that trigger this pathway, it is now known that many other ligands such as il-6 family cytokines, il-10 and neurohormones can also activate proteins in the pathway . The details of early experiments in hematopoietic and other systems with the ifns and cytokines have been summarized in many decent reviews; however, its cardiovascular responses are still limited in literature . In current review, we will mainly discuss the critical role of stat pathway in cardiovascular diseases . Seven stats (stats 1, 2, 3, 4, 5a, 5b and 6) have been identified in mammals and range in size from 750 to 900 amino acids . The structural and functional analysis of these proteins suggests that they have six conserved domains (fig . 1). This includes the n - terminal domain (nh2), the coiled - coiled domain (ccd), the dna binding domain (dbd), the linker domain and the sh2/tyrosine activation domain . In contrast, the carboxyl - terminal transcriptional activation domain (tad) is quite divergent and contributes to stat specificity (fig . 1). The four core domains contact each other by large inter - domain interfaces, suggesting that structural changes induced in one domain may also affect other domains . The identification of stat homologs in simpler eukaryotes suggests that this family arose from a single gene . N - terminal domain; coiled coil domain (ccd); dna binding domain (dbd); linker domain; src homology domain 2 (sh2) and the tyrosine residue (y) phosphorylation sites . The n - terminal half of the protein (~125 amino acids) consists of two relatively poorly characterized domains . This domain is well conserved between these families of protein and is reported to promote cooperativity in dna binding and to regulate nuclear translocation . It represents an independently folded and stable moiety, which can be cleaved from the full - length molecule by limited proteolysis . Several studies suggest that n - terminal dimerization promotes cooperativity of binding to tandem gas (ifn activated sequences) elements . Other studies have suggested that the n - terminal stat domain promotes interaction with the other proteins or receptors and that it regulates nuclear translocation . The coiled - coil domain (amino acids ~135 to ~315) consists of a four - helix bundle that protrudes about 80 laterally from the core structure . Many studies have also implicated the coiled - coil domain in receptor binding and tyrosine phosphorylation in addition to nuclear export . The dna - binding domain (dbd; amino acids ~320 to ~500) recognizes members of the -activated sequence (gas) family of enhancers and (like the upstream coiled - coil domain) appears to regulate nuclear export . The adjacent linker domain (amino acids ~500 to ~600) is important in assuring the appropriate structure of the dna - binding motif and also appears to regulate nuclear export in resting cells . Not surprisingly, the sh2 domain (amino acids ~600 to ~700) is the most highly conserved motif and mediates both receptor - specific recruitment and stat dimerization . The number of direct contact sites between amino acid residues and dna are modest, accounting for a dissociation constant in the nanomolar range . Thus, cooperativity in dna binding is likely to be important in effective transcriptional activity . All of these proteins (stat16) except stat2 are known to homodimerize in vivo . Dimerization requires the binding of a phosphorylated tyrosine activation motif on one stat subunit to the sh2 domain of the other subunit . Finally, the carboxyl - terminus carries a transcriptional activation domain (tad), which is conserved between homologs (e.g., murine and human). However, the carboxyl - terminus varies considerably in both length and sequence between different stat family members . Once again, stat2 is an exception, since its tad sequence diverges considerably between the murine and human homologs . As a part of normal cytokine signaling cascade, the jak - stat pathway is also initiated by binding of a ligand to its receptor in the plasma membrane and the subsequent homo- or heterodimerization of the receptor . In the heart, il-6, il-11, leukemia inhibitory factor (lif), oncostatin m, ciliary neurotrophic factor (cntf) and cardiotrophin - like cytokine (ct-1) are the major cytokines that transduce their signals via glycoprotein 130 (gp130) predominantly to stat3 and 5 . The receptor dimerization, in turn, induces phosphorylation and activation of jak proteins, which are associated with the intracellular domain of the receptor . Jak proteins phosphorylate the receptor, thereby creating docking sites for cytosolic stat proteins via their sh2 domains . Subsequently, stat proteins become phosphorylated on a specific tyrosine residue (for example, tyr705 for stat3) by activated jak kinases, and undergo homo- or heterodimerization by interaction of the phosphotyrosine residue of one stat monomer and the sh2 domain of the other monomer . Once dimerized, these proteins dissociate from the receptor and translocate into the nucleus, where they bind to specific dna sequences and regulate the expression of target genes (fig . 2). In the heart, stat proteins regulate the expression of genes encoding proteins mainly involved in inflammation, cellular signaling, apoptosis, angiogenesis and extracellular matrix composition . The phosphorylation of an additional specific serine residue in the transactivation domain of stat proteins (ser727 for stat3) generally promotes transcriptional activity . The activation and nuclear translocation of stats usually occurs within 15 min, but stat proteins are also rapidly inactivated, resulting in a half - life of nuclear phosphorylated stat between 15 and 30 min . Upon de - phosphorylation by nuclear phosphatases, the jak - stat pathway is not only controlled via phosphorylation of the signaling proteins, but also by negative regulators . Upon activation, stats bind to the promoter region of socs genes (suppressors of cytokine signaling) and upregulate the transcription of these target genes (fig . 2). Socs proteins (mainly socs 1 and 3) negatively regulate the jak - stat pathway by either directly binding to jak, by binding to the receptor and to jak, or by competing with stats for the docking sites at the receptor . Another negative feedback mechanism of the jak / stat pathway comprises shp-2 proteins (src homology 2 domain containing protein tyrosine phosphatase), which dephosphorylate the receptor, jak or stat proteins . The details of the canonical jak - stat signaling pathway have been extensively reviewed elsewhere . These include animal s species, cell type differences and its intra - cellular distribution . Figure 2 . A putative model for jak - stat signaling pathway . Upon binding ligand (il-10), activated stat3 is released from the receptor, dimerize, translocate to the nucleus, and bind with transcription factors to regulate expression of many cardio - protective, anti - inflammatory or growth related genes . As a feedback loop it also regulates expression of suppressor of cytokines signaling genes (socs). In addition to jaks, ptks and mapk, activated by ang ii, may participate in phosphorylation of stat3 . Activated stat3 translocate to the nucleus to activate genes, such as c - fos, c - myc, 2-macroglobulin and tissue inhibitor metalloproteinase-1 (timp-1) by binding to the sis - inducible element (sie) of the promoter . Seven stats (stats 1, 2, 3, 4, 5a, 5b and 6) have been identified in mammals and range in size from 750 to 900 amino acids . The structural and functional analysis of these proteins suggests that they have six conserved domains (fig . 1). This includes the n - terminal domain (nh2), the coiled - coiled domain (ccd), the dna binding domain (dbd), the linker domain and the sh2/tyrosine activation domain . In contrast, the carboxyl - terminal transcriptional activation domain (tad) is quite divergent and contributes to stat specificity (fig . 1). The four core domains contact each other by large inter - domain interfaces, suggesting that structural changes induced in one domain may also affect other domains . The identification of stat homologs in simpler eukaryotes suggests that this family arose from a single gene . N - terminal domain; coiled coil domain (ccd); dna binding domain (dbd); linker domain; src homology domain 2 (sh2) and the tyrosine residue (y) phosphorylation sites . The n - terminal half of the protein (~125 amino acids) consists of two relatively poorly characterized domains . This domain is well conserved between these families of protein and is reported to promote cooperativity in dna binding and to regulate nuclear translocation . It represents an independently folded and stable moiety, which can be cleaved from the full - length molecule by limited proteolysis . Several studies suggest that n - terminal dimerization promotes cooperativity of binding to tandem gas (ifn activated sequences) elements . Other studies have suggested that the n - terminal stat domain promotes interaction with the other proteins or receptors and that it regulates nuclear translocation . The coiled - coil domain (amino acids ~135 to ~315) consists of a four - helix bundle that protrudes about 80 laterally from the core structure . Many studies have also implicated the coiled - coil domain in receptor binding and tyrosine phosphorylation in addition to nuclear export . The dna - binding domain (dbd; amino acids ~320 to ~500) recognizes members of the -activated sequence (gas) family of enhancers and (like the upstream coiled - coil domain) appears to regulate nuclear export . The adjacent linker domain (amino acids ~500 to ~600) is important in assuring the appropriate structure of the dna - binding motif and also appears to regulate nuclear export in resting cells . Not surprisingly, the sh2 domain (amino acids ~600 to ~700) is the most highly conserved motif and mediates both receptor - specific recruitment and stat dimerization . The number of direct contact sites between amino acid residues and dna are modest, accounting for a dissociation constant in the nanomolar range . Thus, cooperativity in dna binding is likely to be important in effective transcriptional activity . All of these proteins (stat16) except stat2 are known to homodimerize in vivo . Dimerization requires the binding of a phosphorylated tyrosine activation motif on one stat subunit to the sh2 domain of the other subunit . Finally, the carboxyl - terminus carries a transcriptional activation domain (tad), which is conserved between homologs (e.g., murine and human). However, the carboxyl - terminus varies considerably in both length and sequence between different stat family members . Once again, stat2 is an exception, since its tad sequence diverges considerably between the murine and human homologs . As a part of normal cytokine signaling cascade, the jak - stat pathway is also initiated by binding of a ligand to its receptor in the plasma membrane and the subsequent homo- or heterodimerization of the receptor . In the heart, il-6, il-11, leukemia inhibitory factor (lif), oncostatin m, ciliary neurotrophic factor (cntf) and cardiotrophin - like cytokine (ct-1) are the major cytokines that transduce their signals via glycoprotein 130 (gp130) predominantly to stat3 and 5 . The receptor dimerization, in turn, induces phosphorylation and activation of jak proteins, which are associated with the intracellular domain of the receptor . Jak proteins phosphorylate the receptor, thereby creating docking sites for cytosolic stat proteins via their sh2 domains . Subsequently, stat proteins become phosphorylated on a specific tyrosine residue (for example, tyr705 for stat3) by activated jak kinases, and undergo homo- or heterodimerization by interaction of the phosphotyrosine residue of one stat monomer and the sh2 domain of the other monomer . Once dimerized, these proteins dissociate from the receptor and translocate into the nucleus, where they bind to specific dna sequences and regulate the expression of target genes (fig . 2). In the heart, stat proteins regulate the expression of genes encoding proteins mainly involved in inflammation, cellular signaling, apoptosis, angiogenesis and extracellular matrix composition . The phosphorylation of an additional specific serine residue in the transactivation domain of stat proteins (ser727 for stat3) generally promotes transcriptional activity . The activation and nuclear translocation of stats usually occurs within 15 min, but stat proteins are also rapidly inactivated, resulting in a half - life of nuclear phosphorylated stat between 15 and 30 min . Upon de - phosphorylation by nuclear phosphatases, stat proteins shuttle back into the cytosol via the nuclear pore . The jak - stat pathway is not only controlled via phosphorylation of the signaling proteins, but also by negative regulators . Upon activation, stats bind to the promoter region of socs genes (suppressors of cytokine signaling) and upregulate the transcription of these target genes (fig . 2). Socs proteins (mainly socs 1 and 3) negatively regulate the jak - stat pathway by either directly binding to jak, by binding to the receptor and to jak, or by competing with stats for the docking sites at the receptor . Another negative feedback mechanism of the jak / stat pathway comprises shp-2 proteins (src homology 2 domain containing protein tyrosine phosphatase), which dephosphorylate the receptor, jak or stat proteins . The details of the canonical jak - stat signaling pathway have been extensively reviewed elsewhere . These include animal s species, cell type differences and its intra - cellular distribution . Figure 2 . A putative model for jak - stat signaling pathway . Upon binding ligand (il-10), activated stat3 is released from the receptor, dimerize, translocate to the nucleus, and bind with transcription factors to regulate expression of many cardio - protective, anti - inflammatory or growth related genes . As a feedback loop it also regulates expression of suppressor of cytokines signaling genes (socs). In addition to jaks, ptks and mapk, activated by ang ii, may participate in phosphorylation of stat3 . Activated stat3 translocate to the nucleus to activate genes, such as c - fos, c - myc, 2-macroglobulin and tissue inhibitor metalloproteinase-1 (timp-1) by binding to the sis - inducible element (sie) of the promoter . All seven stat family members have been reported to be expressed in the heart and/or cultured cardiac myocytes, fibroblasts and endothelial cells . At cellular compartment level, stat localization is not restricted to the cytosol and the nucleus but recently, stat proteins have also been identified in mitochondrial fraction . As for the exact role of jak - stat signaling in cardiac function and diseases, most of the available information relates to stat1 and 3 family members . Various stimuli which activate hypertrophic growth of cardiac myocytes and/or provide cardio - protection have been demonstrated to activate jak - stat signaling in the heart . Importantly, studies have established that these stimuli also enhance cardiac stat functional activity, as assessed by electrophoretic mobility shift assays of dna binding activity, or promoter - reporter assays of transcriptional activity . Mechanical stretch and pressure over - load, myocardial infarction and ang ii treatment has been shown to activate cardiac jak - stat signaling . In addition, ischemia and ischemia / reoxygenation also activates jak - stat signaling in cultured ventricular myocytes, isolated heart preparations or the in situ hearts . It has been well documented that monocytes and macrophages produce inflammatory cytokine to repair the injury during myocardial infarction and hypertrophy . Angiotensin ii also mimics the action of cytokines by activation of tissue inhibitor of metalloproteinase-1 (timp-1), an important factor associated with cardiac remodeling . The mechanisms whereby g - protein receptors couple to tyrosine phosphorylation in general and more specifically to the jak - stat pathway, are not yet clearly elucidated . Previously, it was suggested that yxxq regions of at1 receptors could serve as a docking sites to facilitate stat3 phosphorylation . Alteration in jak - stat signaling pathways were also reported in patients with end - stage dilated cardiomyopathy . The early activation of stat3 during diseased stage could be the protective response of system to reduce the cardiac death and remodeling . Recent reports have shown that transgenic mice with global deletion of stats are embryonically lethal further suggesting its role in development and growth . To sidestep the embryonic lethal phenotype of global stat3 knockout mice and to better understand the rle of stat3 in the heart cardiac myocytes - specific stat3 ko mice have been developed . These mice are significantly more susceptible to cardiac injury under the influence of various stress signals suggesting a protective role of stat3 in heart . However, a major disadvantage of these tissue specific stat3-ko mice is the pathological phenotype, which develops with age and thus limits the direct assessment of the role of stat3 in cardio - protection . Recently, bolli and coworkers generated inducible, cardiac myocyte - specific stat3-deficient mice, which are of great value since they overcome the problems of embryonic lethality and the consequences of chronic alterations in stat3-dependent gene expression . These mice excluded the possibility of age - dependent alterations in apoptosis, fibrosis, capillary density, and cardiac function, and importantly these mice did not show age dependent cardiac hypertrophy or dilatation . Therefore, the inducible cardiac myocyte - specific stat3-ko mouse represents a novel and attractive model to study the role of stat3 in the cardio - protection by ischemic pre- and post - conditioning without the confounding effects associated with chronic stat3 deletion . Mechanism for cardio - protective action of stat3 is not well studied in cardiovascular diseases . Mostly, the protective effect of stat3 in the heart is linked to the reduction in inflammation . Evidence for this notion comes from the studies in which stat3-deficient mice treated with lipopolysaccharide (lps) demonstrated significantly more apoptosis and fibrosis than their wt counterparts . Furthermore, stat3 ko cardiac myocytes secrete significantly more tumor necrosis factor (tnf) in response to lps than those with wt stat3 . The anti - inflammatory and anti - fibrotic effects of stat3 could be due to direct transcriptional inhibition of nuclear factor kappa - b (nfb). Indirectly, we have also shown that use of il-10, an anti - inflammatory pleotropic cytokines, markedly activated stat3 phosphorylation after myocardial infarction injury and thus improve heart function . Many recent studies indicated that stat3 induces the expression of pro - angiogenic factors from resident cardiac cells . It has been shown that overexpression or activation of stat3 in cardiac myocytes enhances vegf expression, which, in turn, promotes myocardial capillary formation . Cardiac myocyte - specific deletion of vegf reduces coronary microvascularization, suggesting that vegf may be an important target gene mediating pro - angiogenic effects of stat3 . A recent interesting study reported that stat3 plays a critical role during pregnancy related adaptive cardiac hypertrophy by activation of angiogenesis (increased vegf) and by inhibition of oxidative stress (increased mnsod level). In our previous report we documented that il-10 markedly increase the vegf and capillary beds in infarct zone after myocardial infarction . This effect of il-10 on microvasculature is stat3-dependent as stat3 inhibitor markedly eliminated the il-10 responses . In contrast, other groups have shown stat3 does not affect the expression of vegf . Using stat3 ko animals they reported that vegf expression is unaffected by stat3 deletion although these animals showed compromised heart function and reduced angiogenesis after myocardial infarction . Interestingly, stat3 ko animals showed increased expression of connective tissue growth factor (ctgf), plasminogen activator inhibitor 1 (pai-1), tissue inhibitor of matrix metalloproteinase 1 (timp1) and thrombospondin 1 (tsp1) compared with wt mice after mi . All these factors are potent suppressor of angiogenesis as well as activator of fibroblast proliferation . In addition, the increased fibrosis and myocyte cell loss might be due to reduced blood and thus oxygen supply to the heart cells in this model . Recently we found that endothelial progenitor cells (epcs) can also play critical role in neovascularization in heart . During ischemic condition (in mi model) both homing and survival of epcs in the heart was markedly reduced . In this fascinating study we have shown that during myocardial infarction the mobilization of epcs from bone marrow (bm) to heart is impaired and contributes to diminished angiogenesis and markedly reduced left ventricular functions . We also found that modulation of inflammation il-10 therapy markedly improves epcs survival and neovascularization in the border zone of the infarct . Interestingly, il-10 dependent increase in epcs survival and function is partially dependent on stat3 signaling, at least in in vitro system . In addition, we also showed that differentiation of functional cardiac myocytes from pluripotent embryonic stem cells (es) under the influence of cytokines and growth factors requires stat3 activity . In this study use of selective stat3 inhibitor, markedly reduced the leukemia inducing factor (lif) and bone morphogenic protein 2 (bmp-2) induced cardiac myocytes differentiation from murine es cells . In addition, sdf1/cxcr signaling is critical for the homing of progenitor cells from bone marrow to the heart for cardiac repair during heart failure . Recently it has been shown that inhibition of stat3 (both pharmacological and genetic knockdown) significantly abolished the sdf1/cxcr1 mediated cardiac protection during ischemia / reperfusion injury . Another pertinent mechanism for cardio - protective action of stat3 is by reducing stress - induced cell death in heart as apoptosis is one of the hallmarks of heart failure . Hydrogen peroxide (a potent cell death activator) treated neonatal rat ventricular myocytes (nrcm) showed reduced expression of phosphorylated stat3 . In isolated rat hearts, perfused with ag-490 (potent jak1/stat3 inhibitor) enhanced the number of apoptotic cardiac myocytes . The anti - apoptotic function of stat3 was also confirmed in mice with a cardiac myocyte - specific deletion of stat3 . These mice showed higher ischemia / reperfusion - induced apoptosis compared with wild type control . Furthermore, the stat3-dependent transcriptional upregulation of anti - apoptotic and cyto - protective proteins (bcl - xl, hsp70 and mnsod) clearly demonstrated the critical role of stat3 in cardiac myocytes survival . Although early activation of stat3 is well established during ischemia / reperfusion (i / r), its transcriptional regulation is probably too slow a process to provide the immediate rescue from cell death during early minutes of reperfusion . Recently, stat3 has been identified in cardiac myocyte mitochondria, and its pharmacological inhibition or genetic ablation impaired complex i respiration and calcium retention capacity . Conversely, a mitochondrial - targeted stat3 overexpression in mice preserved complex i respiration during simulated ex vivo ischemia and reduced the formation of reactive oxygen species . These studies suggest that a pool of stat3 resides in the mitochondria and try to inhibit ros production by inhibition of complex i and ii activity . Evidence for this notion comes from the study where cardiac myocytes specific overexpression of transcriptionally inactive mitochondrial stat3 in transgenic mice hindered the ischemia - induced mitochondrial complete complex i and ii activity, cytochrome c release and ros production . Recent studies, using pharmacological inhibitors, have demonstrated that ischemic post - conditioning preserve mitochondrial complex 1 respiration both in vivo and in vitro . The exact phosphorylation site that is important for the improvement in mitochondrial function by stat3 is not clear but may vary with species: in mouse cardiac myocyte mitochondria serine727 site, whereas both the serine727 and the tyrosine705 site in rat cardiac myocyte mitochondria and only the tyrosine705 site in pigs is deemed important . The increased stat3 phosphorylation at tyrosine705 was associated not only with better preservation of complex 1 respiration but also with improved calcium retention capacity as a measure of mitochondrial permeability transition pore inhibition . The cardio - protective action of stat3 is also partially dependent on the age of animals . In a recent study it has been demonstrated that the beneficial effects of ischemic post - conditioning is lost in aged animals and suggested that reduced stat3 levels may be cause of this effect . Interestingly, in contrast to the cardio - protective nature of active stat3, some recent studies also suggested that the un - phosphorylated stat3 can induce adverse cardiac remodeling . During chronic stress the signals from at1r leads to unregulated expression of stat3 resulting in excessive accumulation of un - phosphorylated stat3 into the nucleus where they can bind with target gene (such as p300/cbp) promoters thereby inducing genes that are involved in adverse heart function . It is believed that the normal mechanisms of stat clearance from nucleus is impaired during chronic stress pathology and thus contribute toward the accumulation of u - stat3 . Moreover, u - stats have been shown to partner with nfb p65 and irf1 in binding to different hybrid dna elements . Hence, u - stat3 may partner with different factors depending on the cell type and sequence context, thereby regulating constitutive gene expression both positively and negatively . Many investigators believe that stats signaling might be detrimental and induce distractive signaling in heart, however, we and others published data indicating that stat3 mediates cardio - protective signaling . Stat3 activation is important for facilitating protective effects in the heart such as compensatory hypertrophy or a reduction in apoptosis . Additionally, as mentioned above stat3 activation is central for the cardio - protection by ischemic pre- and post - conditioning . Stimulation of stat3 activity is expected to be beneficial during the transition from compensated hypertrophy to heart failure . Agonist induced activation of stat signaling in the myocardium could be a potential strategy to induce cardio - protective signaling . However, stats as transcriptional activators or co - activators control the transcription of many target gene and excessive activation of some genes might be detrimental . For example, il-6 mediated excessive activation of inos gene increased stat3-dependent nitric oxide synthesis and decreases cardiac contractility . Therefore, the stat3 activation in cardiovascular disease must be carefully controlled and well defined treatment strategies aiming for a balanced stat3 signaling should be developed in order to protect the heart from pathophysiological stress . In conclusion, stat signaling is an important mechanism in the protection of cardiac cells from different stress . To understand the mechanisms and inhibit the transition of adaptive hypertrophy to maladaptive hypertrophy, stat signaling could play an important role, as it gets activated during physiological hypertrophy and inhibited in chronic pathological conditions . Basic and translational studies should be focused toward understanding the factors / stimuli that regulate stat activity at late stage of diseases . Another area of research that should make a significant stride in the next few years is to generate safer agonists and carrier molecules that can be given to patients with an end - stage disease condition to improve heart function.
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Vulvovaginal candidiasis (vvc) is a common illness attributed to an overgrowth of candida species, and it is estimated that 75% of all women will experience an episode of vvc in their lifetimes . C. albicans accounts for 8095% of all episodes of vvc worldwide [1, 2]. Albicans candida species previously ranged from 5 to 20%; however, the number of reported cases has increased sharply over the last two decades, particularly for cases of c. glabrata [3, 4]. The emergence of resistance may be attributed to the following factors (i) the widespread use of over - the - counter (otc) medications; (ii) long - term use of suppressive azoles; and (iii) the frequent use of courses of antifungal medications [1, 3] or (iv) the increase use of vaginal cultures for reliable diagnoses [2, 5]. There is no evidence to suggest the followings: (i) certain women may be more susceptible to infection by particular candida species over other species, or (ii) there are epidemiologic factors that may predispose women to acute vvc (avvc) versus recurrent vvc (rvvc) [1, 2]. Vvc is also, albeit infrequently, caused by c. parapsilosis, c. tropicalis, and c. krusei [1, 6, 7]. The decreased susceptibility of bloodstream c. krusei isolates to amphotericin b and 5-flucytosine as determined using the broth microdilution method is well documented . However, in vitro susceptibility testing has not been used to evaluate the clinical response of c. krusei vaginitis . In addition, little is known about vaginal c. krusei infections because they are relatively rare . However, c. krusei is known to be inherently resistant to one of the most commonly used antifungal drugs, fluconazole . The signs and symptoms of c. krusei vaginitis appear to be indistinguishable from the signs and symptoms of vvc cases caused by other candida species [6, 10]. Although rare, c. krusei is an intractable cause of rvvc . Furthermore, most institutions have had limited experience with c. krusei vaginitis ., we retrospectively analyzed the epidemiological characteristics of 28 vaginal c. krusei isolates, including host and risk factors . In addition, we investigated the antifungal susceptibility profiles of these isolates to 10 antifungal drugs to determine the most appropriate therapeutic choice(s) in women with c. krusei vaginitis . We examined 1,543 vaginal samples from unrelated women, of which 560 (36.3%) were culture - positive and 983 (63.7%) were culture - negative for candida yeasts, and the medical records of these cases were reviewed . Among the 560 vaginal yeast isolates, c. albicans was the most common species and identified in 242 (43.2%) isolates, followed by c. glabrata in 155 (27.7%), c. krusei in 28 (5.0%), c. kefyr in 20 (3.6%), and in 115 (20.5%) representing several species of candida . The definitions of the clinical presentations of vvc for each group were as follows: avvc (group 1), currently asymptomatic women with initial or sporadic episodes of symptomatic vaginitis, that is, occurring fewer than four times per year (n = 8); rvvc (group 2), symptomatic patients with a history of four or more clinical episodes of vvc per year (n = 13); and controls (group 3), women who incidentally carried a normal level of c. krusei in their vaginal culture without vaginitis, who were completely asymptomatic and had no history of rvvc (n = 7). The control group included a mixed group of asymptomatic women who had no history of rvvc and women who had positive cultures . All participants took part in a short interview, which included questions regarding lifestyle and medical, gynecological, and sexual history . This study was reviewed and approved by the institutional review board at the university of ukurova, adana, turkey . The declaration of helsinki protocols were followed, and the patients provided written informed consent . The c. krusei isolates were recovered on chromagar candida (becton dickinson, heidelberg, germany) and appeared as dull, flat, light mauve to mauve, and colonies with a whitish border . The criteria for the identification of c. krusei were the absence of germ tube production in human serum at 37c at 2 hours, the production of abundant pseudohyphae with some moderate branching on cornmeal - tween 80 agar (difco, detroit, mi, usa), and weak or absent urease activity . These isolates were verified by their assimilation patterns using the api 20c aux method (biomrieux, marcy l'toile, france). C. krusei atcc 6258 was used as a positive control . Antifungal testing was conducted at the department of microbiology, faculty of medicine, gazi university, ankara, using a broth microdilution method and according to the guidelines of the m27-a3 document of the clinical and laboratory standards institute (clsi). Before testing, each isolate was subcultured on sabouraud glucose agar (sga; merck, darmstadt, germany) to ensure purity and viability . The interpretation of antifungal susceptibility was guided by criteria derived from the clsi's m27-a3 protocol . The following antifungal agents were tested: amphotericin b (0.0316 g / ml), 5-flucytosine (0.0664 g / ml), caspofungin (0.0316 g / ml), fluconazole (0.12128 g / ml), itraconazole (0.0316 g / ml), voriconazole (0.00816 g / ml), econazole (0.0078 g / ml), ketoconazole (0.0078 g / ml), miconazole (0.0078 g / ml), and sulconazole (0.0316 g / ml). The minimal inhibitory concentrations (mics) were determined for each antifungal agent and used to classify the susceptibility of the isolates as follows: (i) amphotericin b, mic 1 (g / ml), susceptible (s); (ii) 5-flucytosine, mic 4 (g / ml) s, mic between 8 and 16 (g / ml) intermediate (i), mic 32 (g / ml) resistant (r); (iii) caspofungin, mic 2 (g / ml) r; (iv) fluconazole, mic 8 (g / ml) s, mic between 16 and 32 (g / ml) susceptible dose dependent (s - dd), mic 64 (g / ml) r; (v) itraconazole, mic 0.125 (g / ml) s, mic between 0.25 and 0.5 (g / ml) s - dd, mic 1 (g / ml) r; (vi) voriconazole, 1 (g / ml) s, mic = 2 (g / ml) s - dd, mic 4 (g / ml) r; (vii) ketoconazole, mic 16 (g / ml) r; and (viii) miconazole, mic 4 (g / ml) r . Currently, there are no published criteria for defining econazole and sulconazole susceptibility . These results were expressed in terms of the mic range and the mic50, and mic90 values for each antifungal agent . All c. krusei isolates were declared resistant to fluconazole . C. krusei atcc 6258 and c. parapsilosis atcc 22019 were used as controls, as recommended by the clsi [12, 14]. Continuous variables, such as age and body mass index, were first divided into bins: <30, 3039, 4049, and> 50 years of age and <25 (under or normal weight) and> 25 (overweight or obese) for body mass index . Then, all categorical variables were cross classified by c. krusei infection or carrier status to descriptively summarize the association between the variables using the chi - squared test and to measure the degree of association using the odds ratio with a 95% confidence interval . For the multivariate analysis, logistic regression modeling of the binary data (c. krusei infected, carrier, or neither) was used to determine the significant predictors of c. krusei infection, after adjusting for other factors in the models . Factors with significance levels <0.30 were entered into the multivariate logistic model to determine the significant effect of each factor simultaneously on the prediction of c. krusei infection . C. krusei isolates were recovered from non pregnant patients without diabetes mellitus (n = 9), pregnant patients (n = 6), diabetes mellitus patients (n = 6), and contraceptive user's (n = 7) with no previous history of immunodeficiency who visited the faculty of medicine department of obstetrics and gynecology at ukurova university from 2009 until 2012 . Of the c. krusei isolates, 24 (85.7%) were the only species on plates and four (14.3%) were part of mixed cultures, which were always included by c. albicans . In our group, 24 (85.7%) women were in premenopausal and four (14.3%) in postmenopausal period who had also exposed hormone replacement therapy . The mean age of the women was 40.3 10.4 years (range, 21 to 59 years old). The basic demographic and clinical characteristics of women with vaginal c. krusei isolates are presented in tables 1 and 2 . Perineal laceration is significantly higher (p = 0.006) in the c. krusei group compared with the non - c . Krusei group (table 2). As revealed by multivariate analysis, existence of perineal laceration (p = 0.009) and an age of over 50 years (p = 0.02) the mic results for the control strains of c. krusei atcc 6258 and c. parapsilosis atcc 22019 were within the acceptable range . All the c. krusei isolates were susceptible to amphotericin b, caspofungin, ketoconazole, and miconazole, and 10 of the 28 isolates (35.7%) were defined as s - dd for 5-flucytosine . High mic rates were observed for fluconazole, of which 42.9% of the isolates were s - dd and 57.1% were r. remarkably, only 42.9% and 67.9% of the isolates were susceptible to itraconazole (six s - dd and 10 r) and voriconazole (four s - dd and five r), respectively . The antifungal susceptibilities of c. krusei isolated from patients with avvc and rvvc did not differ significantly from those isolated from the group of women without symptoms of vaginitis (p> 0.05; table 4). To the best of our knowledge, this study is the largest series to exclusively investigate the prevalence of, host and risk factors for, and antifungal susceptibility of the minor isolate c. krusei . We also briefly summarized the baseline and demographic characteristics of women who had c. krusei present in their vaginal samples (tables 1 and 2). Our data suggest that the prevalence of c. krusei is relatively high (5.0%) in this population, displayed no specific host preferences, and was most often associated with rvvc . Perineal laceration and increased age (> 50 years) were significant predictors of c. krusei vaginitis (tables 2 and 3). An important limitation of the present study is the lack of data regarding in vivo therapeutic drug choices and outcomes in c. krusei vaginitis . In addition, the number of women with c. krusei is very small, so, for some of the other factors we examined, the study may not have had sufficient power to detect a difference (tables 13). The presence of mixed cultures may affect the choice of treatment strategy . In our previous study, using chromogenic media, we determined that the percentage of mixed cultures recovered from vaginal samples was as high as 14.1% in adana, turkey . The results of this investigation (14.3%) are similar to those of our earlier study . In addition, our finding that older women (mean age, 40.3 years) are more susceptible to infection corroborates the earlier finding of singh et al . (mean age, 44 years). However, the women studied by singh et al . All had rvvc, whereas our study included not only rvvc cases but also avvc and controls . These authors noted that c. krusei isolates were highly resistant to fluconazole (mic90> 64 g / ml), consistent with our findings . In addition, these authors reported resistance to miconazole (mic90> 4 g / ml), one of the most commonly used otc antifungal agents, which we did not observe . However, in line with our results, clotrimazole was observed to be the most active topical imidazole against c. krusei . Moreover and more importantly, amphotericin b, caspofungin, itraconazole, and voriconazole were demonstrated to have favorable antifungal activity, although, in our study, several strains exhibited obvious high resistance to the latter two drugs . Of note, the authors suggested that the therapy should continue for 26 weeks, regardless of the agent used . On the other hand, a recent study reported that fluconazole - resistant c. albicans appears to be emerging in clinics . Therefore, antifungal susceptibility testing may assist in selecting the appropriate therapeutic drug not only for non - c . Albicans candida vaginitis but also for rare fluconazole - resistant c. albicans vaginitis . In this investigation, amphotericin b, caspofungin, ketoconazole, and miconazole were observed to be active against all c. krusei isolates (table 4). In contrast to the findings of singh et al ., but in line with those of richter et al ., itraconazole exhibited high s - dd and r rates, 35.7% and 21.4%, respectively . Although the new broad - spectrum oral antifungal voriconazole is rarely used in patients with vvc, we observed that 67.9% of the isolates were susceptible to voriconazole . Pfaller et al . Reported a higher rate, stating that 81.5% of 426 genital c. krusei isolates were susceptible to voriconazole using the clsi m44-a disk diffusion method . In conrast to our findings, lyon et al . Reported that fluconazole resistance rates were highly predictive of resistance to voriconazole . Although specific clinical cutoff points have not yet been assigned for econazole and sulconazole susceptibility, we observed low mic values for both drugs . This study is the largest to date to investigate the antifungal drug - resistance profile of c. krusei vaginal isolates and the epidemiologic risk factors of infection . In this investigation, perineal laceration and increasing age (> 50 years) were important predictive factors for c. krusei vaginitis or carrier status (table 3). This study also revealed that the topical imidazoles (ketoconazole and miconazole), which can be prescribed safely in routine practice, were effective against all c. krusei isolates . In addition, the vaginal c. krusei isolates were less susceptible to itraconazole (42.9%) and voriconazole (67.9%) than to other antifungal therapeutics . These findings may have implications for the in vivo therapeutic treatment of c. krusei vaginitis (table 4). Thus, the identification of c. krusei in vaginal samples and in vitro antifungal testing will assist in the selection of appropriate antifungal agents and therapy duration . Future clinical trials to determine the in vivo efficacy of the current drugs for women with c. krusei vaginitis are required.
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Aldehyde oxidases (ec 1.2.3.1) are proteins belonging to the family of molybdo- and tungsten - enzymes, which is represented in both eukaryotes and prokaryotes . In mammals, no tungsten - containing proteins are known, while three other types of molybdo - enzymes, besides aldehyde oxidases--that is, xanthine oxidoreductase (xor), sulphite oxidase (so) and the recently discovered mitochondrial amidoxime reductase component (marc)--have been described . Mammalian catalytically active molybdo - enzymes require a specific form of organic molybdenum, known as the molybdenum cofactor (moco). Unlike that observed in so and marc, the holo - enzymatic forms of aldehyde oxidases and xors contain a sulphurated species of moco [3 - 5]. Aldehyde oxidases and xors are further sub - classified as molybdo - flavoenzymes (mfes), if they require flavin adenine dinucleotide (fad) as a cofactor . Both enzymes are homodimers, with each subunit (approximately 150 kda) consisting of three distinct domains . The 25 kda amino- terminal domain contains two non - identical iron / sulphur clusters, the 40 kda intermediate domain consists of the fad - binding region, while the 85 kda carboxy - terminal domain is characterised by the presence of the substrate - binding pocket, which lies in close proximity to the moco site . One of the main differences between aldehyde oxidases and xors is represented by a nadbinding site, which is absent in aldehyde oxidases . Xors, in their dehydrogenase form, transfer the reducing equivalents generated by the oxidation of the substrate to nad . By contrast, aldehyde oxidases and xors, in their oxidase form, use molecular oxygen as the final electron acceptor, producing hydrogen peroxide . The similarity between aldehyde oxidases and xors is not limited to their general characteristics, and extends to the primary structure . The overall amino acid identity between aldehyde oxidases and xors from the same animal species is approximately 40 per cent . In addition, the primary structures of plant, insect and vertebrate aldehyde oxidases show a remarkable degree of similarity to xors of bacterial origin . This is in line with the idea that the two enzymes are evolutionarily conserved and originated through at least two asynchronous duplication events of the corresponding genes . Some years ago, it was believed that the family of mammalian mfes consisted of only two members--xor and a single aldehyde oxidase (annotated in the national center for biotechnology information [ncbi] database as aox1). The first complete amino acid sequence of a mammalian aldehyde oxidase was deduced from the molecular cloning of the corresponding bovine cdna in our laboratory . Based on the high level of amino acid similarity, it soon became apparent to us that this sequence corresponded to a human cdna that had been originally identified as xor . The first hint of the presence of multiple aldehyde oxidases in certain animal species came from an early analysis of the limited number of mouse expressed sequence tags (ests) available in the ncbi database . This allowed us to identify and isolate two other mouse cdnas encoding catalytically active proteins highly related to bovine, mouse and rat aox1, the primary structure of which had been elucidated in the meantime [16 - 18]. In a subsequent study, we identified a fourth murine functional enzyme and demonstrated that rats are characterised by the same complement of four aldehyde oxidases as mice . Each novel rodent aldehyde oxidase was shown to be the product of distinct loci, mapping with the original aox1 orthologous genes to a small region (approximately 350 kilobases) of mouse chromosome 1 and rat chromosome 9 . The three novel proteins present in rodents were named aldehyde oxidase homologues 1, 2 and 3 (aoh1, aoh2 and aoh3), and the corresponding genes were referred to as aoh1, aoh2 and aoh3, respectively . The current nomenclature adopted by the ncbi is different, as aoh1 is referred to as aox3, while aoh2 and aoh3 have been renamed aox4 and aox3l1, respectively (table 1). We will conform to this nomenclature throughout the paper, and refer to the various aldehyde oxidases present in different mammalian species as aox1, aox3, aox4 and aox3l1 . The term' aldehyde oxidase(s)' will be used in a general sense, whenever no distinction between the various isoforms of the family is meant . The accession numbers of the proteins or predicted translation products of the corresponding genes present in the genbank or ensembl databases are indicated in the rightmost column . The official or proposed gene symbol (' gene symbol') along with the acronym originally suggested in our previous references (' our original symbol') are also indicated . * at present, it is unclear whether macaca mulatta aox4 is a functional gene or a pseudogene . The dendrogram shown in figure 1 summarises our current knowledge of the phylogenesis of aldehyde oxidases . The primary structure of these proteins is relatively well conserved throughout evolution, and the most primitive organism showing evidence for one such enzyme is the flat worm, caenorhabditis elegans . A rooted dendrogram was obtained by the phylip method after clustal - w computer - aided alignment of the indicated proteins . Poere aox1 = poecilia reticulata (guppy) aox1; coeel aox1/aox2 = caenorhabditis elegans aox1 and aox2; drome aox1/aox2/aox3/aox4 = drosophila melanogaster aox1, aox2, aox3 and aox4; arath aox1/aox2/aox3/aox4 = arabidopsis thaliana aox1, aox2, aox3 and aox4; lyces aox1/aox2/aox3 = lycopersicon esculentum (tomato) aox1, aox2 and aox3; zeama aox1/aox2 = zea mays aox1 and aox2; xenla aox1 = xenopus laevis aox1; gorgo aox1 = gorilla gorilla aox1 . Bona fide aldehyde oxidase homologous proteins do not seem to be represented in prokaryotes, although many molybdenum and tungsten enzymes with low levels of amino acid similarity to vertebrate aldehyde oxidases are known (eg see blase' et al . ). This is at variance with what has been observed for xor, the ancestors of which can be easily traced back to prokaryotes and very primitive eukaryotes, like aspergillus nidulans . As already discussed, aldehyde oxidases and xors are highly related proteins and have a common origin . Aldehyde oxidases are indeed the products of duplication events from an ancestral eukaryotic xor gene; however, two structurally different families of aldehyde oxidases can be recognised on the basis of the amino acid sequences (figure 1). A first cluster consists of nematode, insect and plant enzymes, while a second group contains all vertebrate aldehyde oxidases . Vertebrate aldehyde oxidases are closer to xors than to aldehyde oxidases from more primitive animals and plants . This finding is suggestive of two separate and evolutionarily divergent duplication events from an ancestral xor gene . The first event produced the aldehyde oxidase precursor to worm, insect and plant enzymes . This is supported by the intron / exon structure of all the vertebrate aldehyde oxidase and xor genes, which are strikingly conserved and much more complex than those of homologues from the lower eukaryotes and plants . The two original duplications were followed by a number of other such events that led to the extant complement of aldehyde oxidase genes in the plant and animal kingdoms . The evolution of vertebrate aldehyde oxidases (figure 2) is characterised by a first phase of asynchronous gene multiplication events, which started in certain birds . Fishes (danio rerio) and amphibians (xenopus laevis) are endowed with a single functional aldehyde oxidase gene . The figure contains a schematic representation of the aldehyde oxidase genes in selected vertebrates for which complete or almost complete genomic sequence data are available . The chromosomal location is indicated on the right (nd, not determined). Whenever the structure of the gene is predicted solely on the basis of the genomic sequence, and the corresponding cdnas have not been characterised, the genbank locus number (loc) is indicated . At present, it is unclear whether macaca mulatta aox4 is a functional gene or a pseudogene . The corresponding gene products show the highest level of amino acid identity to the rodent aox1 isoenzyme, supporting an orthologous relationship . D. rerio or x. laevis aox1 has the conserved 35/36 exon structure typical of all vertebrate aldehyde oxidase genes . In addition, fish aox1 and xor map to different chromosomes, which is also characteristic of all vertebrates except primates . Some avians (chicken) show evidence of a gene duplication event involving aox1 and resulting in the production of a new synthenic gene (on chromosome 7), which we named aldehyde oxidase homologue (aoh). Aoh is characterised by an identical exon / intron structure, with perfect conservation of exon / intron junctions along the coding sequence . The amino acid sequences of chicken aox1 and aoh protein products are approximately 60 per cent identical and only more distantly related to the corresponding xor enzyme (40 per cent identity). The presence of two aldehyde oxidase genes in birds does not seem to be a general phenomenon . In fact, a blast search indicates that the zebra finch, taeniopygia guttata, has a single aox1 locus on chromosome 7 (table 1 and figure 2). Moving up along the evolutionary ladder to marsupials (monodelphis domestica, opossum), the exon - intron structure of all the genes is strictly conserved, indicative of two further gene duplication events . Three of the loci (aox1, aox3 and aox4) map to chromosome 1, whereas the fourth gene (aox3l1) is located on chromosome 7 . The genes identified in m. domestica are the orthologues of the four loci present in rodents (mouse and rats). The evolutionary process of the aldehyde oxidase gene cluster in mammals is characterised by a sudden and species - specific shift from multiplication to suppression / deletion . Bos taurus (cow) seems to have maintained three active aldehyde oxidase genes (aox1, aox4 and aox3l1) on chromosome 2 . The absence of nucleotide sequences with similarity to aox3 strongly suggests that this gene has been deleted . Deletion of the aox3 gene seems to be a conserved feature in another herbivore, the horse, although our present view of the aldehyde oxidase cluster in this animal species is still incomplete . The genome of dogs is characterised by two seemingly active aox4 and aox3l1 loci and two inactive aox1 and aox3 pseudogenes clustering on chromosome 37 . The vestiges of numerous exons with nucleotide similarity to the rodent aox1 and aox3 genes are easily identified on two separate regions slightly upstream of the dog aox4 and aox3l1 loci . It is interesting to notice that the dog is currently the only mammalian species that seems to be lacking aox1, in addition to aox4 . This observation has important implications, as this mammal is devoid of aldehyde oxidase activity in the liver . Humans are endowed with a single functional aldehyde oxidase gene, namely aox1, consisting of the canonical 35 conserved coding exons . This is the result of the persistence of the aox3 deletion and the simultaneous transformation of aox4 and aox3l1 into inactive, albeit transcribed, pseudogenes . Aox1 and the two pseudogenes map to a short segment on chromosome 2q . Functional inactivation of the aox4 and aox3l1 genes occurred before the appearance of the human species, as chimpanzees (pan troglodytes) are endowed with the same complement of aldehyde oxidase genes and pseudogenes as humans . Functional suppression of aox4 and aox3l1 seems to be the result of two recent, distinct and asynchronous events, based on the results obtained in the old - world monkeys (macaca fascicularis and macaca mulatta). In fact, the genomes of these monkeys seem to contain two functional (aox1 and aox3l1) as well as one or two inactive (aox3 and aox4) pseudogenes clustering on chromosome 12 (m. terao, unpublished result). In summary, two phylogenetically distinct branches of the evolutionary process led to the extant complement of worm / insect / plant and vertebrate aldehyde oxidases through an equivalent number of primitive and distinct gene duplication events involving the xor ancestor . These initial events were followed by a divergent series of species - specific and more recent gene multiplication events that occurred not only in vertebrates but also in plants, worms and insects . Maize, tomato and arabidopsis thaliana are respectively characterised by two, three and four structurally very similar aldehyde oxidase genes . Similarly, the flatworm, c. elegans, and the fruitfly, drosophila melanogaster, are endowed, respectively, with two and four aldehyde oxidases . The most ancient member of the vertebrate aldehyde oxidase family is aox1 ., the evolutionary order of appearance of aox3, aox4 and aox3l1 is unknown; however, a number of considerations led us to propose that aox4 is more ancient than aox3, which, in turn, appeared earlier than aox3l1 . The data available on the tissue distribution of human aox1 are limited [23 - 25]. The richest source of the enzyme is the liver, however, where aox1 serves an important role in the metabolism of xenobiotics . Aox1 is also present in the respiratory system, where it can be identified in the epithelial cells lining the trachea and bronchi, as well as in the alveolar cells of the lung . In the digestive system, the kidney is another source of aox1, with synthesis of the protein occurring in the proximal distal and collecting tubules . Finally, the prostate and adrenal glands seem to contain detectable amounts of the protein . The localisation of aox1 in the central nervous system is largely unknown, although it was reported that the corresponding transcript is expressed in the glial component of the anterior horns of the spinal cord . In general, the tissue distributions of aox1 in humans and baboons, the only other primate for which studies are available, are concordant, particularly in relation to the presence of the enzyme in the livers and the respiratory systems . Two mouse aldehyde oxidases (aox1 and aox3) have an overlapping tissue distribution, which is largely superimposable with that of human aox1 . While this is expected in the case of aox1, given its orthologous relationship with the human enzyme, the observation is important in the case of aox3 . By far the richest source of the two enzymes is the liver, where aox1 and aox3 are present exclusively in the cytosolic fraction of the hepatocyte compartment . Different mouse strains are characterised by different relative amounts of the two hepatic enzymes, however . High levels of aox3 and much lower levels of aox1 are present in the outbred cd1 and the inbred c57bl/6j strains . By contrast, the two inbred strains dba/2 and cba have an almost complete deficit of expression of aox3 . This deficit is the result of epigenetic silencing of the corresponding gene by methylation of the regulatory sequences . Significant expression of both aox1 and aox3 is observed also in the lung, which is the second richest source . Limited amounts of aox1 and aox3 are also believed to be present in the central nervous system, on the basis of in situ hybridisation experiments http://www.brainatlas.org / aba/. Expression is limited to the choroid plexus, which is the organ devoted to the production and re - absorption of the cephalorachidian fluid, and the motor neuronal cell population in the brain and spinal cord . While cellular co - localisation of aox3 in the choroid plexus is clear, similar evidence is not available for the motor neurones . Low levels of aox1 and/or aox3 are also inferred from the expression data available in the online mendelian inheritance in man (omim) section of the ncbi website http://http./www.ncbi.nlm.nih.gov/. Of particular interest is the presence of aox1 in the skin (which was recently confirmed), the mammary gland and the genitourinary tract . The richest source of aox4 is the harderian gland, a prominent intra - orbital exocrine gland involved in diverse homeostatic functions, such as thermoregulation, lubrication of the eye surface, control of pheromonal cues and regulation of the circadian rhythm [30 - 32]. The gland is present in various vertebrate species, but is absent in humans and primates . Far lower amounts of aox4 have also been identified in the epidermal layer of the skin and the keratinised epithelia lining the oral cavity, oesophagus and proximal portion of the stomach . In the skin, the epidermis is not the only structure containing aox4, as sebaceous glands are also enriched in the corresponding mrna . Interestingly, sebaceous and harderian glands share common characteristics, producing a lipid - rich secretion, which is involved in thermoregulation and pheromone release . Finally, aox3l1 is unique, being limited to the secretory cells of the bowman's gland, the most important exocrine gland of the olfactory mucosa . These structures are present in all vertebrates, including humans, with the exception of fish . The serous / mucous product of bowman's glands is important in shaping the microenvironment necessary for a proper stimulation of the nasal neuroepithelium by odorants . Bowman's glands are so rich in aox3l1 that they were used as the source for the purification of the enzyme to homogeneity . Most of the literature on aldehyde oxidases has focused on this aspect of their biology [33 - 38]. All the members of the family are characterised by broad substrate specificity, oxidising numerous types of molecule . In spite of their name, aldehyde oxidases catalyse not only the oxidation of aldehydes into the corresponding carboxylic acid, but they also hydroxylate n - heterocyclic molecules with high efficiency . Human aox1 and rodent aox1/aox3 may actually represent the major cytosolic enzymes metabolising xenobiotics in the liver . The enzymes are involved in the phase i metabolism of numerous compounds of both medical and toxicological relevance, potentially acting in concert with the microsomal cytochrome p450 system [33 - 39]. Extensive discussion of the compounds metabolised by human or rodent aldehyde oxidases is beyond the scope of this paper, and the reader is referred to specific reviews for more details . It is worth mentioning that aldehyde oxidases have an important role in the metabolism of the anti - tumour and immunosuppressive agents, methotrexate, 6-mercaptopurine and azathioprine, and aldophosphamide, the active metabolite of cyclophosphamide . Aldehyde oxidases have also been reported to metabolise the antimalarial agent, quinine and the anti - viral drug famcyclovir . Finally, human aox1 is known to cause the inactivation of the hypnotic, zaleplon . As evident from this short and incomplete list, the drug metabolising activity of aldehyde oxidases is predominantly related to the ability of these enzymes to hydroxylate n - heterocyclic rings . From a toxicological perspective, the aldehyde oxidase activity present in mammalian liver has been reported to play a major role in the oxidation of environmental pollutants such as phthalazines . By contrast, it is unlikely that mouse aox1 and aox3 are of any relevance for the oxidation of the ethanol metabolite, acetaldehyde, into acetic acid . Although a wealth of data are available on exogenous substrates, endogenous substrates of aldehyde oxidases are still being sought . The kyoto encyclopedia of genes and genomes (kegg, http://www.kegg.jp) contains a number of metabolic pathways listing potential physiological substrates of aldehyde oxidases, such as the serotonin metabolite, 5-hydroxyindoleacetaldehyde (pathway: ko00380), and the amino acid catabolites (s)-methylmalonate semi - aldehyde (pathway: ko00280) and gentisate aldehyde (pathway: ko00350). At present, however, there is no direct evidence of the significance of human aox1 or any of the other mammalian homologues in the oxidation of these substrates . Aldehyde oxidases are also purported to play a role in the metabolism of vitamins b3 (nicotinamide), b6 (pyridoxal phosphate) and a (retinol). The vitamin b3 metabolite, n1-methylnicotinamide is a good substrate for semi - purified human and monkey aox1 preparations . Pyridoxal, the vitamin b6 precursor, is oxidised to 4-pyridoxic acid by the human enzyme and by the two mouse liver aldehyde oxidases, aox1 and aox3 (terao m., unpublished results). Pyridoxal is currently the sole example of a substrate that shows a certain degree of selectivity for a specific aldehyde oxidase protein as it is not recognised by purified mouse aox4 . Vitamin a metabolism is probably the endogenous pathway for which more stringent information on the involvement of aldehyde oxidases is available . The ability of mammalian aldehyde oxidases to catalyse the oxidation of retinaldehyde (a physiological precursor) into retinoic acid (the active metabolite of vitamin a) was discovered in rabbit liver cytosol and confirmed using purified preparations of mouse liver aox1 . In our hands, retinaldehyde has been one of the best substrates not only of mouse aox1, but also of aox3, aox4 and aox3l1 . The km and vmax measured for the purified forms of aox1, aox3 and aox4 compare well with the same parameters reported for the three aldehyde dehydrogenases, aldh1a1, aldh1a2 and aldh1a3, which have long been known to catalyse the oxidation of retinaldehyde and to play a critical role in the morphogenetic activity of retinoic acid during the development of the vertebrate embryo . Support for the relevance of aox4 for the local synthesis and accumulation of retinoic acid in the harderian gland has recently been provided by the phenotypic, genetic and biochemical characterisation of the first aldehyde oxidase knockout mouse to be generated . Although a substantial amount of information on the structure and evolution of mammalian aldehyde oxidases is available, little is known about the physiological function of these enzymes in mammals, with particular reference to humans and relevant animal models . Progress in establishing the significance of this class of enzymes for the homeostasis of the mammalian organism is clearly a priority . To this end, it will be necessary to identify the endogenous substrate(s) of human aox1 and all the other mammalian isoenzymes . This is likely to require an integrated approach based on the definition of the substrate(s) using purified enzyme preparations, and validation of the results in vivo . It is envisaged that in vivo studies in humans will require the measurement of relevant molecules in biological fluids, using cohorts of individuals genotyped for the presence of aox1 allelic variants with alterations in the catalytic activity of the corresponding protein products . It will therefore be important to increase the number of population studies designed to discover new aox1 single nucleotide polymorphisms (snps) (see, for instance, our reference rs41309768, in the single nucleotide polymorphism database [dbsnp] section of the ncbi database). Similar studies will also have to be performed in experimental animals, with the caveat that mice and rats are characterised by the presence of multiple aldehyde oxidase forms, as discussed above . In this context, strains of mice characterised by a different profile of aldehyde oxidases, as well as specific knockout animals, will represent useful tools to this end . Uplc / q - tof - ms / ms, followed by chemometrics and analysis of principle components, would very likely identify one or more metabolic pathways that are perturbed in the absence of each one of the mouse aox enzymes . Association studies aimed at defining the relevance of aox1 for the aetio - pathogenesis and progression of specific human diseases are also needed, as they may provide insights into the functional significance of the enzyme . These studies will integrate the knowledge that is likely to be generated from the phenotypic analysis of the genetically engineered mice that are already available or will be available in the near future . Along these lines, our studies on the aox4 knockout mouse have already provided evidence that the corresponding protein is involved in the control of skin homeostasis and in the postnatal development of the harderian gland . Whether the two phenomena are linked to a decrease in the local metabolism of retinaldehyde to retinoic further knowledge is likely to be gained soon by phenotypic analysis of the newly generated aox3l1 knockout animal (m. terao, unpublished results). Nevertheless, the data already gathered strongly suggest that the various aldehyde oxidases are unlikely to play a vital function in the homeostasis of the mouse, as both types of knockout animal develop normally and are fertile . Three final considerations are worth making . The first two concern, again, the basic problem of the physiological significance of aldehyde oxidases . In this regard, a key point relates to the reason(s) why the evolution of vertebrate organisms is associated with a first wave of multiplication and a subsequent phase of deletion / suppression of the aldehyde oxidase genes . These phenomena are likely to be related to the necessity for developing specific and possibly tissue - related functions in certain animal species, which must have become dispensable in humans and primates . The idea is readily acceptable for an enzyme like aox4, which is highly enriched in the harderian gland, as the structure is absent in humans and primates . It may be also viable in the case of aox3l1, if the protein serves a function in the recognition of odorants . Indeed, it is well known that olfaction is much more developed and sophisticated in rodents than in humans . The presence of an extra enzyme (aox3) in the liver of the mouse, relative to the human, is more complicated to explain . Nevertheless, in generating hypotheses as to the real function of the various aldehyde oxidases, an open mind should be always kept, since the function of these proteins may not necessarily be related to their enzymatic activity . The third consideration is of a more practical nature and is relevant for the role exerted by aldehyde oxidases in drug metabolism . With respect to this, it is clear from what has been reported here that caution should be exercised in using rodents, dogs and possibly rhesus monkeys as good proxies for the human situation . This work was made possible by unrestricted grants from the fondazione monzino, the associazione italian per la ricerca contro il cancro (airc) and the fondo di investimento per la ricerca di base (firb). We would also like to thank mr f. deceglie and mr a. soave for the artwork.
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Paul ehrlich described mast cells for the first time more than 100 years ago, through their characteristic metachromatic staining with aniline dyes . He noticed that these cells had abundant and dense granules; he assumed that the granules were caused by excessive feeding . These cells were abundant in areas near tumors, so ehrlich postulated that they played a role in the protection against cancer . Since then, many functions were proposed for mast cells in the regulation of organism homeostasis, but experimental evidence to support these assumptions was lacking . The discovery of mice with a diminished number of mast cells, and their reconstitution with bone marrow - derived mast cells, provided a tool to test these hypotheses and to extend our knowledge about the functions of mast cells in the body . Mast cells derive from hematopoietic precursors that are released from the bone marrow into the bloodstream and are recruited to connective tissues, where they complete their differentiation . Mast cells can live for months in the tissues, where they proliferate in response to different signals . The tissues that usually contain abundant mast cells are those that interact with the external environment (the skin and the gastrointestinal, urogenital and respiratory tracts), where mast cells are located near lymphatics and blood vessels, or close to nerve terminals or smooth muscle cells . The morphology of mast cells depends on their anatomical location, ranging from rounded - shaped to elongated forms, when they are associated with blood vessels . Their size ranges from 5 to 20 m and their cytoplasm contains abundant granules, with approximately 1,000 granules per cell . Mast cell granules contain several chemical mediators, including histamine, proteoglycans, cytokines like tnf- and il-4, and neutral proteases like tryptase, chymase, carboxypeptidase and cathepsin g. granule composition is not identical in all mast cells: human mast cells that reside in the skin have all the aforementioned neutral proteases, while epithelial lung mast cells have significant amounts of tryptase, but lack chymase, carboxypeptidase and cathepsin g. accordingly, human mast cells are classified as either mct (skin) or mt (lung). In addition to these pre - formed mediators that are stored in granules, mast cells are efficient producers of lipid - derived mediators, such as platelet - activating factor (paf) and the arachidonic acid - derived prostaglandins, thromboxanes and leukotrienes . Mast cells are also efficient producers of many soluble protein mediators, such as cytokines, chemokines and antimicrobial peptides [6, 7]. Activation of mast cells induces the release of one group of these mediators or of all of them, depending on the triggering signal . However, not all the mediators are released at the same time: chemical mediators from the granules are secreted in seconds after stimulation, while lipid - derived mediators are produced minutes after activation and protein soluble mediators are detected hours after the initial trigger . Mast cells produce abundant histamine, and since the discovery that histamine is a crucial element for the development of type i hypersensitivity, the relevance of mast cells for the initiation of allergic reactions was underscored . By far, the most studied mast cell - activating signal is the cross - linking of fcri by ige and allergen, an event that leads to mast cell degranulation and to the release of several chemical mediators that cause the immediate inflammatory response characteristic of type i hypersensitivity . As a consequence, much of our knowledge of mast cell biology is biased towards their participation in this pathological process . Type i hypersensitivity, or allergic inflammation, is an acute inflammation produced in sensitized individuals when they are exposed to the corresponding allergen . Sensitization occurs when dendritic cells capture an allergen in the airways, migrate to the regional lymph nodes and activate nave t cells . In the presence of il-4 (derived from mast cells, eosinophils or innate lymphoid cells), these cytokines promote class - switch recombination in b cells, which then produce allergen - specific ige . Ige binds to fcri on mast cells, which are then primed for activation upon re - exposure to the allergen . During this subsequent exposure, the allergen cross - links fcri - bound ige, which leads to mast cell activation and degranulation . The mediators derived from activated mast cells cause bronchoconstriction, vasodilation, increased vascular permeability, mucus production and recruitment of eosinophils and neutrophils . Repetitive or persistent exposure to the allergen leads to a chronic allergic inflammation, which causes tissue injury and remodeling; this last process includes the severe narrowing of the airway lumen observed in asthmatic patients . However, mast cells are conserved in different groups of vertebrates, and from an evolutionary point of view, it makes no sense that humans should have conserved a cell population that causes a harmful and even deadly response if it conferred no evolutionary advantage . So, in the last years, the study of mast cells has been redirected to investigate their participation in processes such as innate immune response to pathogens, regulation of inflammation, wound healing and regulation of tumoral growth . In this review, we focus on the mechanisms used by mast cells in the lung to keep the proper function of this vital organ . Mast cells have a vast collection of cell membrane receptors that allow them to sense the external environment . A set of these receptors are pattern recognition receptors (prr) that recognize pathogen associated molecular patterns (pamp). These receptors are crucial for the initiation of the innate immune response, and they are classified in five groups: toll - like receptors (tlr), nod - like receptors (nlr), c - type lectin receptor (clr), rig - i - like receptor (rir) and scavenger receptors . Mast cells express at least one receptor of each group, allowing them to recognize a multitude of microorganisms, including viruses, bacteria, protozoa, fungi and multicellular parasites [13 - 23] (fig . 1). The first evidence that mast cells are crucial elements of the immune response to pathogens came from a study of the infection of the rat gastrointestinal tract with multicellular parasites . There are few studies that investigate if mast cells play a role in the containment of parasitic diseases in the lungs . One of the firs studies that established a role for mast cells in fungal infections in vivo reported that, in a mouse model of infection with sporothrix schenckii, mast cell depletion decreased the severity of cutaneous lesions . Whether mast cells in the lungs plays a role in fungal infections needs to be evaluated, but in vitro experiments suggest that they do have a role, because they are activated by aspergillus fumigates . Identified for the first time that mast cells are essential for the early containment of bacterial infections in the lungs; klebsiella pneumonie, a gram - negative bacteria that causes community - acquired pneumonia, was among the bacteria tested in this study . The authors observed that the lung bacterial load was higher in mice deficient in mast cells, compared with wild - type mice, and this correlated with a defective recruitment of neutrophils to the lung . These results indicated that mast cells did not have a direct antimicrobial activity, but they induced inflammation through tnf-, which allowed the recruitment of neutrophils . Mast cells also participate in the immune response to mycoplasma pneumoniae, a bacteria that is among the smallest free - living organisms and lacks a classical bacterial cell wall . Airway infection of mice deficient in mast cells induced faster weight loss, more severe pneumonia, higher mortality and increased bacterial burden in the lungs, compared with wild - type mice . This work showed that mast cells play an important role in innate immune response against bacteria that infect lungs . Tularemia is a life - threatening disease caused by francisella tularensis, a gram - negative coccobacillus that can be spread through aerosols . Intranasal infection of wild - type mice with f. tularensis induces an increased accumulation of mast cells in the lungs . Accordingly, mast cell - deficient mice showed impaired bacterial control and a significant decrease in il-4 production . In fact, mast cells confer protection by inhibiting bacterial entry and replication in macrophages, thus showing a different mechanism by which mast cells confer protection against bacterial infection in the lungs . It is caused by mycobacterium tuberculosis (mtb), a bacteria that has a cell wall unique among bacteria, and that infects and usually persists in the lungs . Studies with humans have shown an increase of mast cells in the lymph nodes of patients with tuberculous lymphadenitis . In vitro studies demonstrated that mtb activates mast cells, inducing degranulation, production of il-6 and tnf- and even bacterial internalization through lipid rafts [36, 37]. Moreover, in vivo experiments have suggested a role of mast cells in the containment of mtb infection . In one of the first studies, mice received c48/80, a compound that induces mast cell degranulation, before infection with mtb . These mice showed an altered production of cytokines and chemokines, which induced a decrease in lung inflammation and an increase in mtb load, compared with untreated mice . A second study used tlr-2 knockout mice, which are highly susceptible to mtb infection . When these mice received mast cells that express tlr-2 before infection with mtb, they had an improved inflammatory response, with myeloid cell recruitment and granuloma formation that correlated with increased protection against the bacilli . These studies reinforce the notion of mast cells as crucial players of the lung innate immune response against bacterial pathogens . Cytomegalovirus is a beta - herpes virus that causes interstitial pneumonia in immunocompromised patients and is a serious problem in patients with an organ transplant . Infection of mast cell - deficient mice with cytomegalovirus is followed by high viral replication and delayed virus clearance from the lungs, compared with wild - type mice . Interestingly, this phenomenon was associated with a defective production of the chemokine ccl5 (also known as rantes) and a decreased recruitment of cd8 + t cells into the infected lungs, providing evidence that mast cells can recognize viral infection and induce the recruitment of essential effector cells to the site of infection in the lung . Influenza is a frequent infectious disease of the human respiratory tract, which is caused by a group of viruses from the orthomyxoviridae family; the most common of these viruses is influenza a. these viruses have attracted attention because they are associated with pandemics that have severely affected human health, like the spanish flu of 1918 . Interestingly, studies with this strain have reported alterations in lung architecture, which are caused by an excessive inflammatory response promoted by an uncontrolled production of inflammatory cytokines and chemokines, also known as cytokine storm . In a mouse model of infection with a pathogenic strain of h5n1 virus, lung lesions were reduced if mice were treated with ketotifen, a well - known mast cell degranulation inhibitor . Moreover, mice survival was dramatically increased if mice received a combined treatment of ketotifen and oseltamivir, an antiviral drug . These results suggest that mast cell activation correlated with pathology development during h5n1 infection, and that inhibition of mast cell activation improved host health . Recently showed that the pathology associated with influenza a virus infection is significantly decreased in mast cell - deficient mice, compared with wild - type mice . This event is associated with a lower production of the inflammatory cytokine tnf- and the chemokines ccl2, ccl3, ccl4, cxcl2 and cxcl10 in the lungs, suggesting a critical role of mast cells during the induction of the cytokine storm . The studies in mice described above show that mast cells play a pro - inflammatory role against bacterial and viral infections, with both beneficial and detrimental effects . On the one hand, mast cells are critical for the innate immune response because they recruit effector cells that are fundamental for the containment of the pathogens during the early stages of infection; this event has been observed in other organs besides the lungs . On the other hand, excessive mast cell activation by pathogens can lead to damage and loss of lung function, as evinced by influenza studies . This excessive mast cell activation has also been observed in calves with paroxystic respiratory distress syndrome caused by bovine respiratory syncitial virus . Recent evidence indicates that mast cells also play a predominant role in the development of pathology during dengue virus infection . St john a, et al . Observed a direct correlation between the serum levels of chymase and the severity of dengue disease in humans; interestingly, treatment of mice with drugs that block mast cell degranulation or with an antibody that blocks leukotriene receptors improved the vasculopathy associated with dengue virus infection . These studies point to mast cells as a new therapeutic target to limit the damage provoked by excessive inflammation during infectious diseases, such as influenza and dengue . In contrast with their classical pro - inflammatory function, it has been reported that mast cells can produce significant amounts of anti - inflammatory cytokines, like il-10 . Mast cell - produced il-10 plays a crucial role in limiting the sterile inflammation provoked by allergic contact dermatitis or uv radiation . Moreover, mast cell - produced il-10 is essential for the induction of immune suppression by uv exposure [49, 50]. Recently demonstrated that mast cells produce il-10 during in vivo bladder infection with uropathogenic escherichia coli, and this event was necessary to maintain the tolerance that allowed bacterial persistence . This study demonstrated that microorganisms can tune mast cells into a pro - inflammatory or an anti - inflammatory program in order to subvert the immune response . Choi et al, recently showed that salmonella typhimurium injects a potent phosphatase (sptp) into the cytosol of mast cells; sptp interferes with mast cell activation, even with a very strong signal (fcri cross - linking). This strategy is also exploited by yersinia pestis, which uses a phosphatase (yoph) with high homology to sptp to dampen mast cell activation . Pathogenic bacteria are not the only ones that effectively suppress mast cell activation, symbiotic bacteria have established a delicate balance with the immune response in order to colonize and survive in different anatomical locations of the host . Breakdown of such balance usually results in the development of different pathologies . So, it is not surprising that bacteria that usually colonize the gastrointestinal tract can regulate mast cell activation . One of the first evidences of this strategy came from a study in which murine mast cells were stimulated with non - pathogenic e. coli from human gut . Unexpectedly, mast cells did not degranulate, even after incubation with a high bacterial load, which contrast with the swift degranulation in response to pathogenic e. coli . Furthermore, mast cells incubated with non - pathogenic e. coli lost the ability to degranulate in response to strong activators, such as a23187 and fcri cross - linking . A similar phenomenon is also observed with lactobacillus and bifidobacterium, other probiotic and symbiotic bacteria that reside in the human gut [56, 57]. These results are of great interest in view of the association that exists between the use of probiotics and the prevention and treatment of several atopic diseases . However, how this prevention and treatment are achieved is still a matter of discussion, and several mechanisms have been proposed to explain this phenomenon . Recent evidence derived from a mouse model of allergic inflammation points out that probiotics have the ability to decrease the effector phase in allergic diseases, even in the presence of specific ige antibodies . This effect is related to the inhibition of mast cell activation through the alteration of the signaling pathways triggered by fcri . The probiotics bifidobacterium breve and lactobacillus rhamnosus have been proposed as treatments for atopic diseases because of their potent effect in suppressing mucosal mast cell degranulation during chronic asthma . As described in the previous section, mast cells play an essential role in the initiation of the innate immune response to pathogens . In fact, all the cells that participate in type i hypersensitivity also participate in type 2 immune responses, which protect against infections with bacteria, viruses and multicellular parasites, and participate in the clearance of venoms, xenobiotics and irritants .type 2 innate lymphoid cells and th2 lymphocytes produce il-5, which induces eosinophil activation and recruitment; they also produce il-9, which causes basophil and mast cell proliferation, and il-13, which increases mucus production and causes fibrosis . These cell populations constantly interact with each other: for example, type 2 innate lymphoid cells produce il-13 in response to mast cell - derived prostaglandin d2, for which they have a specific receptor, crth2 . Since mast cells play a fundamental role during the early stages of type i hypersensitivity and also during the initiation of the innate immune response to pathogens, two important questions arise: 1 . Is the response of mast cells to pathogens affected in an allergic environment? 2 . Can pathogens exacerbate type i hypersensitivity through mast cell activation? In relation to the first question, there is considerable epidemiological evidence that asthma is a risk factor for viral and bacterial infections in the lungs, but what promotes this increased susceptibility to infections is poorly understood [63 - 67]. A deficient immune response could explain this increased susceptibility; for example, jung j et al . Observed that asthmatic patients have decreased titers of specific pneumococcal antibodies when compared to not - asthmatic individuals . Other authors suggest a defective innate immune response as the cause of this increase in the susceptibility to infections . Several mechanisms of the innate immune response are altered in allergic patients, including a defective epithelial barrier, an excessive mucus production and a decreased production of cytokines . However, this defective innate immune response apparently does not affect the potential of mast cell to combat infections, as is demonstrated in a mouse model of infection with mycoplasma pneumonie in an allergic setting . In relation to the second question, infectious diseases, especially those caused by bacteria and viruses, are considered important environmental elements that exacerbate atopic diseases . Several reports indicate that infections with bacteria and viruses that affect the respiratory tract induce the production of ige antibodies specific for bacterial or viral components . Infection of newborn mice with respiratory syncital virus (rsv) induces the production of virus - specific ige, and reinfection of these mice caused the airway symptoms classically associated with an allergic response . Moreover, in a mouse model of acute infection with influenza a virus, an enhanced susceptibility of the host to develop a severe allergic response to house dust - mite was described . These studies suggest that the induction and the exacerbation of allergic responses by pathogens are associated with a modification of the environment that favors ige production; however, another mechanism could be the exacerbation of mast cell responses . As we discussed earlier, several bacterial and viral pathogens can induce mast cell activation, inducing a pro - inflammatory environment that recruits effector immune cells . But how does a mast cell respond if it is simultaneously activated through fcri and through receptors that sense infection? Early studies suggested that this double stimuli results in an increased activation of mast cells . For example, mast cell lines activated through fcri and infected with rhinovirus release more histamine and produce more cytokines than mast cell lines activated with a single stimuli . Patients with allergic rhinitis that were experimentally infected with rhinovirus showed increased levels of histamine and eosinphil recruitment in bronchoalveolar lavages, compared with allergic individuals that were not infected with rhinovirus, suggesting an altered mast cell response . Several reports also associate infection with pathogenic bacteria with the development of an environment that facilitates or exacerbates the development of type i hypersensitivity . For example, the pathogenic bacteria mycoplasma pneumoniae, streptococcus pneumoniae, haemophilus influenza and chlamydia pneumoniae induce the production of ige specific for bacterial components, suggesting that infection promotes an environment favorable for the development of allergies [76 - 78]. However, little information exists about how these pathogenic bacteria could affect mast cell activation through fcri . As mentioned above, the only bacteria that have been analyzed in this respect are symbiotic bacteria from the gastrointestinal tract (e. coli, lactobacillus spp and bifidobacterium spp), and these bacteria ameliorated fcri - mediated activation of mast cells, showing a therapeutic potential of these bacteria for allergic reactions in the gut . However, other symbiotic bacteria that colonize a different environment, the vagina, apparently do not mimic this effect . Interestingly, new evidence has demonstrated that the human respiratory tract is home to several symbiotic bacteria, and environments that were thought to be sterile, like the bronchial tree, in fact contain about 2,000 bacterial genomes per cm . Moreover, asthmatic patients showed altered bacterial communities in their airways, when compared with healthy individuals, with an increase in haemophillus spp and a decrease in prevotella spp . How these changes in microbiota airway affect the progress of type i hypersensitivity and how they alter mast cell function are fields that need to be clarified in the future . This process develops in an orderly fashion, involving hemostasis, inflammation, cell proliferation and remodeling of the affected tissue . Alterations in the sequence, duration or intensity of any of these stages result in an impaired tissue repair that is usually reflected in the development of different pathologies . Several cell populations are involved in this process, and cells of the immune system have been implied in different steps of the process . First, they tend to accumulate and degranulate in the wounded tissue, favoring the recruitment of effector cells, mainly neutrophils and macrophages . The neutral proteases chymase and tryptase favor the degradation of the extracellular matrix [83, 84]; mast cell production of arachidonic acid - derivatives induces the production of type i collagen by fibroblasts, and mast cell production of fibroblast growth factor (fgf), vascular endothelial growth factor (vegf) and transforming - growth factor 1 (tgf-1) is considered important for fibroblast proliferation and for the formation of new blood vessels . However, much of the knowledge about the role of mast cells during wound healing comes from studies in the skin, and little work has been performed in the lung, perhaps because of the lack of an appropriate model of lung injury, making this an interesting field that needs more research to clarify the role of mast cells in this environment . Paul ehrlich described that mast cells were abundant in areas near tumors, and assumed that these cells played an important role in the regulation of tumor growth more than 100 years ago . Since then, several reports have confirmed that mast cells usually accumulate in areas near tumors, and that this accumulation is associated with a poor prognosis in breast cancer, melanoma, hodgkin s lymphoma, oral squamous cell carcinoma, renal carcinoma, pancreatic cancer, endometrial cancer, cervical cancer, etc . In 2000, imada et al . Observed that patients with stage i non - small cell lung adenocarcinoma had an increased number of mast cells in tumor areas around blood vessels, and those patients with higher mast cells counts had a worse prognosis . It has been suggested that tumor cells produce chemotactic mediators that attract mast cells to the tumor, but few of these chemotactic mediators have been identified . One example is stem cell factor (scf), which is produced by several tumor - derived cell lines, including a human alveolar adenocarcinoma cell line . Scf interacts with the receptor c - kit, which is expressed on mast cells, and directs their migration to the tumor . How mast cells contribute to tumor growth is still matter of research, but three main mechanisms have been proposed: 1) production of tumor growth factors, 2) facilitation of tumor angiogenesis and 3) modification of the interaction between the epithelial cells and the stroma . A mast cell product that leads to the proliferation of tumor cells is histamine, which promotes the proliferation of alveolar basal carcinoma cells . Adrenomedullin - stimulated mast cells produce cytokines, such as fibroblast growth factor (fgf) and vascular endothelial growth factor (vegf), which are associated with increased angiogenesis in vivo . Observed an increase in the number of mast cells that produce vegf in patients with lung adenocarcinoma . In contrast with previous reports by several authors, welsh et al . Reported that, in non - small cell lung adenocarcinoma, the number of mast cells in the tumor stroma had no correlation with tumor progression, while an increased number of mast cells in the tumor cell islets correlated with an improved prognosis . These results suggested an anti - tumor role for the infiltrating mast cells, but the mechanism was not investigated, and further studies are needed to clarify this issue . Mast cells can trigger different mechanisms that contribute to the homeostasis and adequate function of the lungs . Disequilibrium in the function of mast cells can lead to pathological states, of which asthma is the most recognized . Mast cells play an essential role in type i hypersensitivity (allergic inflammation) and also during the early stages of the innate immune response to pathogens . Several causes, from infections to tumor growth, alter the function of mast cells (fig . The regulation of mast cell activation by conventional drugs or microorganisms, either pathogenic or symbiotic, opens a new window of opportunities to develop new treatments that could preserve and restore lung health.
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Tsao et al ., estimated that 2040% of patients with cancer will develop bms during the course of their illness . More than 80% of bms are detected after the primary tumor has been diagnosed (metachronous metastases) and less frequently, they are the first manifestation of disease or are diagnosed at the same time as the primary tumor (synchronous metastases). Schouten affirmed that literature data reported that, the 5 years cumulative incidence of bms was approximately 16%, 10%, 7%, 5%, and 1% for lung cancer, renal cell cancer, melanoma, breast cancer, and colorectal carcinoma, respectively . Due to the advanced nature of disease at the time of presentation in patients with intracranial metastases, particularly if symptomatic, treatment options have been limited . According to borgelt et al ., all patients with bms typically receive corticosteroids, which will improve edema and neurologic symptoms in approximately 70% of patients, nonetheless, the median survival with steroids alone is approximately 2 months . The irradiation of the whole brain radiotherapy (wbrt) remains the most commonly undertaken treatment for multiple metastases and is associated with increases in the median survival, compared with steroids alone, to approximately 46 months . Because of wbrt leads to serious neurologic or neuro - cognitive deficits (motion and eloquio reduction), in selected subgroup of patients this technique must be discussed in relation to other therapeutic alternatives such as stereotactic radiotherapy (srt) and radiosurgery (srs) or the use of new chemotherapy drugs . However, for patients with short life expectancy and poor performance status, supportive care alone may be the more appropriate oncologic approach . In according to data published on 2012 from international practice survey on the management of bms different treatment schedules are administered in wbrt, particularly 30 gy in 10 daily fraction is most used in usa and europe while 20 gy in five daily fractions is preferred canada and australia / new zeeland . Survival prediction model that might lead decision making when choosing between best supportive care and wbrt were developed . The aim of our retrospective study was to analyze the outcomes of patients with newly diagnosis of multiple bms referred to our institution for wbrt, in order to identify the prognostic factors useful for clinical identification of a subgroup that really benefits from radiation therapy . From january 2010 to february 2014, 80 patients with diagnosis of bms, not eligible for srt or srs, underwent wbrt at our institution . In this preliminary study, were retrospective anlyzed 36 medical records of patients, with a mean age of 63 years (range from 30 to 79), selected because they had performed a radiation treatment comparable in terms of contouring, dose, and fractionation . With the intent of detecting several prognostic factors useful to identify patients who really benefit from wbrt compared to those to be addressed to supportive care alone, were examined all the following clinical and radiological parameters: gender, age (50 vs. 5160 vs. 6170 vs.> 70 years), karnofsky performance status (kps) (kps <70 vs. kps 70), number of bms on computed tomography (ct) and/or magnetic resonance images (5 and> 5), presence or absence of perilesional edema, presence or absence of necrosis pattern, and histology of primary tumor [table 1]. In our retrospective study, these data were extrapolated from computerized medical records, which were recorded by the radiation oncologist at the time of the medical examination . Characteristic of patients as a retrospective study, the ethics committee approval was not required . Although crucial in the choice of treatment strategy, predicting accurately the length of survival of bms cancer patients is difficult because systemic therapies often led patients survival beyond estimated survival rates . Steroid therapy was administrated in all patients along wbrt course, with a tailoring dosage in relation to the neurological symptoms and signs reported before, during, and after treatment . Patients will be immobilized in the supine position using an individually customized thermoplastic face mask (klarity medical and equipment co. ltd ., guangzhou, china). For treatment planning a noncontrast - enhanced ct for virtual simulation (acqsim, philips, the netherlands) was performed, including whole brain down to the inferior plate of the ii cervical vertebral body . Images were transferred to a treatment planning system (oncentra masterplan v3.3, sp 3, nucletron, elekta, atlanta, ga, usa). Planning target volume (ptv) was generated adding 5 mm margin to the whole brain clinical target volume . The selected organ at risks such as a lens, optic nerves, eyeballs, and spinal cord were outlined . Wbrt was administered using two opposite shaped latero - lateral fields with 45 collimator rotation . The field - in - filed technique was implemented in order to reduce the percentage of ptv receiving more than 110% (v 110%) and 107% (v 107%) prescribed dose, as well as dmax . A dose of 2.53 gy was delivered with six megavoltage machine, 5 days / week, for 1210 fractions for a total dose of 30 gy . All patients alive at the time of analysis were censored with the date of last follow - up . Survival was calculated from the 1 day after the end of wbrt . Statistical univariate analysis (kaplan - meier method and log - rank test) and multivariate analysis (cox hazards proportional model) were performed in order to identify prognostic factors influencing survival in case of wbrt administration . Nonsmall cell lung cancer (nsclc) represented the most common histological type (47.5%) followed by breast cancer (22%) [table 1]. The 6, 12, and 20 months overall survival (os) rate were 47%, 19%, and 17%, respectively . Median overall survival time in our clinical records, patients with breast cancer had a better median survival of 9 months compared with 3.5 months for nsclc . On univariate analysis, high kps score, absence of perilesional edema and the number of bms were <5 were significant prognostic factors correlated with os (p = 0.0003, p = 0.03 and p = 0.037, respectively) [figures 24 and table 2]. Karnofsky performance status and overall survival presence / absence of edema around metastatic lesions and overall survival correlation the number of brain metastases on radiological images and overall survival relation univariate analysis of survival on multivariate analysis, performed with cox regression, of all potential prognostic factors for os only kps has been confirmed statistically significant (p = 0.006), while a trend was showed for breast cancer primary tumor (p = 0.098) [table 3 and figure 5]. Multivariate analysis of survival os in relation to histology of primary tumor during the treatment course, 44.4% of patients had a tolerable toxicity profile whereas 28% experienced symptomatic intracranial hypertension syndrome and neurological deficits that required inpatients supportive setting . After wbrt, the most frequents adverse events were headache and dizziness (25% of incidence in both cases) whereas 25% of treated patients showed no symptomatic effects . Only two patients discontinued definitively steroid therapy after wbrt . Finally, nevertheless the radiation dose to the scalp ranged between 28 and 31 gy in all patients, in four cases we registered . Hair regrowth after 3 months, from the end of wbrt probably, due to individual biological parameters . Patients with bms are a heterogeneous population and treatment depends on the individual clinical setting . There is a lack of high - quality randomized evidence to clarify the value of wbrt versus supportive care alone in patients with bms, while no guidelines are available for clinicians to identify which subsets of patients should be managed with supportive care alone, without wbrt . The aim of this retrospective study was to assess the effectiveness of wbrt in adult patients with newly diagnosed bms and to individuate potential prognostic factors useful to drive therapeutic choice that is, wbrt versus supportive care . In the present study, the limitations of our preliminary study are related to inadequate consideration about the information on the status of systemic disease at the moment of wbrt and the extensive range of time in which patients were retrospectively enrolled . Historically, wbrt has been utilized as the main treatment modality for the management of bms . The addition of wbrt to steroids extended median survival to 36 months, but in the last decade there has been mounting evidence regarding the toxic effects of wbrt, especially serious neurocognitive impairments . Therefore, the clinical indication of wbrt must be the result of the appropriate balance between the potential benefits and possible sequelae, taking into account the clinical implementation of modern radiation therapy approaches, such as srs or hippocampal - avoidance wbrt, and patients performance status, toward a more tailored and disease - specific treatment management . Numerous factors in addition to the presence of active systemic disease have been correlated with poor patient survival: kps <70 poor neurologic status, number of bms, age, time from primary to bms, and tumor histology . In patients affected with bms, literature data show that histology of primary tumor must be considered in order to optimize the therapeutic choice, because there are histological subtypes associated with more favorable os, such as breast cancer versus others more radio - resistant correlated with poor patient survival, such as lung cancer or melanoma . In particular, to date, diagnosis of central nervous system involvement in breast cancer patients is becoming more common because of treatment strategies are improving with a longer os . In order to individualized a subgroup of patients that benefit from wbrt, gaspar et al ., analyzed 1200 patients from three consecutive radiation therapy oncology group (rtog) trials, which tested several different wbrt fractionation schemes and radiation sensitizers, using the rtog recursive partitioning analysis (rpa) prognostic system . A prognostic subgroup of patients was identified according to age at diagnosis, absence or presence of extracranial disease, kps score and status of primary cancer . On the basis of this analysis, the median survival of patients with bms ranges from 2.3 to 7.1 months, opening the debate regarding whether the patients in the best prognosis category should or should not be treated with more aggressive therapies to control intracranial disease . However, the rpa classification failed to correctly predict long - term survival in the majority of patients . In a recent publication confirmed the importance of performance status assessment in the therapeutic decision . In patients with widely disseminated cancer and short life expectancy, wbrt did not significantly improve survival compared with supportive palliative care alone . By contrast, in patients with the extra cranial controlled systemic disease, good performance status, and a limited number of bms wbrt may be a beneficial therapeutic option both for the quality of life and survival . Multivariate analysis of our preliminary data shows that the kps correlates with significant evidence to a better prognosis while a statistically significant trend is highlighted for breast cancer, and this is in agreement with literature data . Regarding the number of bms was not detected statistical significance, as it could be seen from the literature, perhaps for the small power of our sample . Knowledge of prognostic factors of patients with bms is crucial for appropriate therapeutic choice and our preliminary study indicate that, even if in this heterogeneous group of few patients, kps and breast cancer primary tumor are parameters useful to help clinicians to individualize well - selected patients with good life expectancy, in according with literature . Especially kps, beyond the small number of patients of our preliminary study was anyway an important prognostic factor, immediate and easily available, useful in daily clinical practice everywhere . Whole brain conformal radiotherapy continues to be an efficacious treatment in the management of bms . There is clear recognition that not all bms patients that undergo wbrt have survival outcome equivalently . Patients with poor performance status, extensive brain disease, and unfavorable primary tumor histology have less evident beneficial effects from wbrt and probably are best managed with supportive care alone . Taking into account specific prognostic factors, in our preliminary, high kps, and breast cancer primary tumor seem to characterize a subgroup of patients with favorable prognosis.
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A frequent and difficult challenge in women s reproductive health relates to the decline in reproductive outcome and the associated increase in chromosomal aberrations with increased female age . The centers for disease control (cdc) in atlanta provides a comprehensive annual report that includes the results for 90% of the infertility clinics in usa . For the year 2005, the average live - birth rate for in vitro fertilization (ivf) and fresh embryo transfer procedures was 34% . The live - birth rate sharply declined with age, from 43% in women aged <35 years to 6% in women aged> 42 years.1 the decline in live - birth rate reflects a decline in response to ovarian stimulation, reduced embryo quantity and quality, decreased implantation rates, and an increased incidence of miscarriages and fetal aneuploidy . The lack of an age - related decline in pregnancy outcome when donor oocytes are used suggests that the target of the reduced fecundity with aging is the oocyte . Moreover, there is a well - established association between late maternal age and an increased risk for oocyte and embryo aneuploidy.2,3 unlike other cells, the mammalian oocyte and early embryo are completely dependent on mitochondrial oxidative phosphorylation (oxphos) for their supply of energy.4 there is an increased rate of consumption of atp in the mature oocyte that is essential for its ability to undergo normal fertilization . Therefore, it is possible that as women and their oocytes age, oocyte mitochondrial function may decrease influencing many of the processes of oocyte maturation, especially nuclear spindle activity and chromosomal segregation, resulting in an increased rate of aneuploidy.5,6 this hypothesis is supported by data demonstrating that mitochondrial mutations in follicular cells increase with age suggesting that oxphos and atp production in the follicle are impaired in older women.7 it has been demonstrated that embryo implantation potential is correlated with the atp content of the embryo.8 wilding et al9 used radiometric fluorescence techniques to measure changes in mitochondrial inner membrane potential (m) of two - to - three - day - old fresh human embryos . The above authors found a strong correlation between low m and the age of the patient as well as a state of random segregation of chromosomes between the blastomeres, likely resulting from the presence of an abnormal meiotic spindle . The embryos that had chaotic mosaics were significantly slower in their rate of cleavage and significantly more common among the older group of patients . We initially sought to determine whether it is possible to improve oocyte mitochondrial function of elderly gravidas using mitochondrial nutrients . This is a group of endogenous vitamins that were previously shown to directly or indirectly protect mitochondria from oxidative damage . The best studied of these nutrients to date are alpha - lipoic acid (r - ala), coenzyme q10 (coq10), and resveratrol, all of which have been shown to have numerous benefits in neurodegenerative and cardiovascular diseases.1013 we used an aged mouse model comparing retired breeders, eight - month olds, and mice randomized to receive either coq10, resveratrol, r - ala or vehicle alone for a period of 18 weeks before superovulation . Compared to aged controls, coq10 treatment significantly increased the number of ovulated oocytes, increased oocyte mitochondrial atp production, and reduced ros levels to levels comparable to those detected in oocytes of young animals.14 coq10 is a lipid soluble electron transporter that transports electrons from complexes i and ii to complex iii in the mitochondrial respiratory chain and is essential for the stability and action of complex iii.15 it also participates in the transport of protons in the mitochondria to maintain the membrane potential and drive atp formation through atp synthetase.16 coq10 is a major cellular antioxidant.17 there is a gradual, age - related decline in the tissue levels of coq10.18 mutations of genes involved in coq10 synthesis may lead to coq10 deficiency, characterized by clinical disorders involving mitochondrial dysfunction in the nervous system, skeletal muscles, and endocrine glands.19 one study examined the use of coq10 in the in vitro culture of bovine embryos and found a superior rate of early embryo cleavage, blastocyst formation, percentage of expanding blastocysts, and a larger inner cell mass . These changes were associated with an increased atp content in the group of embryos cultured with coq10 . Other investigators have demonstrated an increased atp production in lymphocytes supplemented with coq10.20 as a result of this preliminary mouse study, we decided to conduct a comparative human study . We hypothesized that the administration of coq10 two months before and during an ivf intra cytoplasmic sperm injection (icsi) cycle will improve oocyte energy production and reduce free oxygen radicals, thus resulting in a lower rate of oocyte aneuploidy . The aim of the study was to compare the post - meiotic rate of aneuploidy using polar body biopsy and comparative genomic hybridization (cgh) array for all chromosomes among patients treated with coq10 and placebo . The study was a double blind, placebo - controlled, randomized trial recruiting patients from two sites and using a single lab (toronto center for advanced reproductive technology toronto, ethics review board (reb #08 - 0205-a). The trial was registered on the clinical trials website (www.clinicaltrials.gov) (nct01048385). Body mass index (bmi)> 38 kg / m early follicular phase (day 24) serum follicle stimulating hormone (fsh) level> 20 miu / ml abnormal uterine cavity as evidenced by sonohysterogram or hysterosalpingography any current use of systemic steroid medication or any infertility treatment within three months of study enrollment any contraindication to being pregnant and carrying a pregnancy to term contraindication for the use of coq10, superfact, menopur, hcg, estrase, and progesterone suppositories any ovarian or abdominal abnormality that may interfere with adequate tvs evaluation absence of one or two ovaries clinically relevant systemic disease (eg insulin - dependent diabetes, adrenal dysfunction, organic intracranial lesion, polycystic ovarian syndrome, hyperprolactinemia, or hypothalamic tumor) or serious illness (neoplasia) history (within past 12 months) or current abuse of alcohol or drugs administration of any investigational drugs within three months before the study enrollment any medical condition that may interfere with the absorption, distribution, metabolism, or excretion of the study drugs; gastrointestinal diseases; mal absorption syndromes; and liver dysfunction unexplained gynecological bleeding ejaculated sperm not sufficient for icsi abnormal controlled ovarian hyperstimulation (coh) screening blood done for both partners, including prolactin, thyroid stimulating hormone, hiv serology, hepatitis b and c serology, rubella, group and screen, and syphilis serology before participation in the study and the concurrent use of any of the following drugs: daunorubicin, doxorubicin, blood pressure medications, warfarin, timolol, atorvastatin, cerivastatin, lovastatin, pravastatin, simvastatin gemfibrozil, tricyclic antidepressant medications (including amitriptyline, amoxapine, clomipramine, desipramine, doxepin, imipramine, nortriptyline, protriptyline, and trimipramine), multivitamins, or any vitamin supplementation except folic acid . Patients were assigned in chronological order according to the day of study enrollment to a computer - generated randomization . Each enrolled participant received a pre - assigned package containing either placebo or coq10 for the duration of the study . Randomization was done following the signing of the informed consent . In the first cycle, each patient received six capsules of micronized coq10 (100 mg softgels) or placebo (softgels and gelatin capsules containing rice oil and starch, respectively) (aor advanced orthomolecular research inc ., 19 st ne calgary, ab, canada, nhpd registration code 135307). Study participants took 600 mg of coq10 once a day with breakfast, orally, or identical placebo capsules for up to three cycles if pregnancy did not occur . All subjects took either coq10 or placebo for two months . On day 3 of the following cycle, they started ovarian stimulation for ivf while continuing the consumption of the supplements . Coh was performed using the short microdose gnrh agonist flare protocol (smf) or the long mid - luteal gnrh agonist protocol (lmg) according to the discretion of the primary physician . All gonadotropins used for this study were human menopausal gonadotropin (hmg) (identical batch of menopur, ferring pharmaceuticals, toronto, on, canada) and subcutaneous (sc) buserelin acetate 0.05 mg twice daily for the microdose flare protocol and 0.2 mg daily for the long luteal protocol (suprefact, sanofi - aventis canada inc, laval, qc, canada), and continued until the day before human chorionic gonadotropin (hcg) administration (10,000 units; pregnyl, merck, kirkland, qc, canada). Both drugs were continued daily until follicular development was considered adequate (at least three follicles> 17 mm and the e2 level acceptable for the number of follicles present). Oocyte retrieval was done 36 hours following the hcg injection under ultrasound guidance and with the use of local anesthesia and mild sedation . The oocytes were fertilized with icsi with no regard to the quality of the sperm to prevent contamination of the polar body biopsy sample with sperm dna . The biopsied polar bodies were then sent to mount sinai services (mss genetic lab, toronto, ca, usa) for dna extraction and whole genome amplification followed by array cgh, a microarray - based cgh to analyze copy number of all 24 chromosomes (bluegnome ltd, breaks house, mill court, great shelford, cambridge cb22 5ld, uk). The results of the polar body chromosome analysis were available 24 hours after the biopsy to serve for embryo selection and allow either day 3 or day 5 embryo transfer . One to three embryos were transferred mostly on day 3 post - retrieval when available . The rest of the viable embryos were frozen for subsequent embryo transfer in another cycle if needed . Luteal support consisted of 100 mg of progesterone suppositories (kingsway pharmacy, toronto, on, canada) vaginally three times a day starting on the day of embryo transfer and continued for two weeks until the serum beta hcg result . If pregnant, the subjects stopped coq10/placebo and luteal support continued until 10 weeks of gestation . The primary outcome measure was the number of euploid eggs per retrieval, and secondary outcome measures included cumulative pregnancy rate per retrieval and cumulative live - birth rate per retrieval . Statistical analysis was done using graphpad (prism version 5.00 for windows, graphpad software, san diego, ca, usa, www.graphpad.com). Power analysis was calculated using the following assumptions: alpha 0.01, 90% power, background oocyte aneuploidy for the chosen group of patients80%, the intervention will result in a 25% reduction in aneuploidy rate, and an expected average of six mature oocytes per retrieval in this group of patients . Based on these assumptions, we found that a total of 54 patients would be needed for this two - treatment parallel - design study . Parametric data were analyzed using unpaired t - test with welch s correction for unequal distribution . The trial was registered on the clinical trials website (www.clinicaltrials.gov) (nct01048385). Body mass index (bmi)> 38 kg / m early follicular phase (day 24) serum follicle stimulating hormone (fsh) level> 20 miu / ml abnormal uterine cavity as evidenced by sonohysterogram or hysterosalpingography any current use of systemic steroid medication or any infertility treatment within three months of study enrollment any contraindication to being pregnant and carrying a pregnancy to term contraindication for the use of coq10, superfact, menopur, hcg, estrase, and progesterone suppositories any ovarian or abdominal abnormality that may interfere with adequate tvs evaluation absence of one or two ovaries clinically relevant systemic disease (eg insulin - dependent diabetes, adrenal dysfunction, organic intracranial lesion, polycystic ovarian syndrome, hyperprolactinemia, or hypothalamic tumor) or serious illness (neoplasia) history (within past 12 months) or current abuse of alcohol or drugs administration of any investigational drugs within three months before the study enrollment any medical condition that may interfere with the absorption, distribution, metabolism, or excretion of the study drugs; gastrointestinal diseases; mal absorption syndromes; and liver dysfunction unexplained gynecological bleeding ejaculated sperm not sufficient for icsi abnormal controlled ovarian hyperstimulation (coh) screening blood done for both partners, including prolactin, thyroid stimulating hormone, hiv serology, hepatitis b and c serology, rubella, group and screen, and syphilis serology before participation in the study and the concurrent use of any of the following drugs: daunorubicin, doxorubicin, blood pressure medications, warfarin, timolol, atorvastatin, cerivastatin, lovastatin, pravastatin, simvastatin gemfibrozil, tricyclic antidepressant medications (including amitriptyline, amoxapine, clomipramine, desipramine, doxepin, imipramine, nortriptyline, protriptyline, and trimipramine), multivitamins, or any vitamin supplementation except folic acid . Patients were assigned in chronological order according to the day of study enrollment to a computer - generated randomization . Each enrolled participant received a pre - assigned package containing either placebo or coq10 for the duration of the study . Randomization was done following the signing of the informed consent . In the first cycle, each patient received six capsules of micronized coq10 (100 mg softgels) or placebo (softgels and gelatin capsules containing rice oil and starch, respectively) (aor advanced orthomolecular research inc ., 19 st ne calgary, ab, canada, nhpd registration code 135307). Study participants took 600 mg of coq10 once a day with breakfast, orally, or identical placebo capsules for up to three cycles if pregnancy did not occur . All subjects took either coq10 or placebo for two months . On day 3 of the following cycle, they started ovarian stimulation for ivf while continuing the consumption of the supplements . Coh was performed using the short microdose gnrh agonist flare protocol (smf) or the long mid - luteal gnrh agonist protocol (lmg) according to the discretion of the primary physician . All gonadotropins used for this study were human menopausal gonadotropin (hmg) (identical batch of menopur, ferring pharmaceuticals, toronto, on, canada) and subcutaneous (sc) buserelin acetate 0.05 mg twice daily for the microdose flare protocol and 0.2 mg daily for the long luteal protocol (suprefact, sanofi - aventis canada inc, laval, qc, canada), and continued until the day before human chorionic gonadotropin (hcg) administration (10,000 units; pregnyl, merck, kirkland, qc, canada). Both drugs were continued daily until follicular development was considered adequate (at least three follicles> 17 mm and the e2 level acceptable for the number of follicles present). Oocyte retrieval was done 36 hours following the hcg injection under ultrasound guidance and with the use of local anesthesia and mild sedation . The oocytes were fertilized with icsi with no regard to the quality of the sperm to prevent contamination of the polar body biopsy sample with sperm dna . The biopsied polar bodies were then sent to mount sinai services (mss genetic lab, toronto, ca, usa) for dna extraction and whole genome amplification followed by array cgh, a microarray - based cgh to analyze copy number of all 24 chromosomes (bluegnome ltd, breaks house, mill court, great shelford, cambridge cb22 5ld, uk). The results of the polar body chromosome analysis were available 24 hours after the biopsy to serve for embryo selection and allow either day 3 or day 5 embryo transfer . One to three embryos were transferred mostly on day 3 post - retrieval when available . The rest of the viable embryos were frozen for subsequent embryo transfer in another cycle if needed . Luteal support consisted of 100 mg of progesterone suppositories (kingsway pharmacy, toronto, on, canada) vaginally three times a day starting on the day of embryo transfer and continued for two weeks until the serum beta hcg result . If pregnant, the subjects stopped coq10/placebo and luteal support continued until 10 weeks of gestation . The primary outcome measure was the number of euploid eggs per retrieval, and secondary outcome measures included cumulative pregnancy rate per retrieval and cumulative live - birth rate per retrieval . Statistical analysis was done using graphpad (prism version 5.00 for windows, graphpad software, san diego, ca, usa, www.graphpad.com). Power analysis was calculated using the following assumptions: alpha 0.01, 90% power, background oocyte aneuploidy for the chosen group of patients80%, the intervention will result in a 25% reduction in aneuploidy rate, and an expected average of six mature oocytes per retrieval in this group of patients . Based on these assumptions, we found that a total of 54 patients would be needed for this two - treatment parallel - design study . Parametric data were analyzed using unpaired t - test with welch s correction for unequal distribution . Patient enrollment to the study began in 2010 and was continued until 2012 . In may 2012, levin et al had published a paper describing the negative effects of polar body biopsy on embryogenesis.21 the authors compared the rate of cleavage and fragmentation among 145 embryos that underwent a polar body biopsy and 276 that did not . Their results showed a significant rate of cleavage arrest and fragmentation in the biopsied embryos . These findings corresponded with our own experience showing a much poorer embryo quality for the biopsied embryos . We therefore decided to terminate the study before reaching the target number of enrolled patients . At the point the study was terminated (june 2012), we had recruited a total of 39 patients who were evaluated and randomized (17 to the coq10 group and 22 to the placebo group). Only 27 had started the treatment with the supplements (12 of the coq10 group and 15 of the placebo group). In all, 24 patients completed the treatment cycle and had a polar body biopsy (pbbx) and embryo transfer done (10 of the coq10 group and 14 of the placebo group). Six patients withdrew from the study for personal reasons, three for conceiving spontaneously, two for poor compliance, one for failing to achieve the target bmi, and three because of poor ovarian response . The average age of the patients was 39.1 and 39.0 for the placebo and coq10 groups, respectively . No differences were found in day 3 of fsh, day of hcg administration, and the total number of hmg units administered . In the coq10 group, 30.8% of the patients were treated with the long luteal gnrh agonist protocol, whereas in the placebo group 7.7% . The rest of the patients in both groups were treated with the short microdose flare protocol (table 1). Pmol / l for the coq10 group and 6875 pmol / l for the placebo group . The percentage of top quality embryos at 48 and 72 hours was 81.4 and 64.7% for the coq10 group, and 66.0 and 42% for the placebo group, respectively (table 2). A total of 114 zygotes underwent pbbx, dna extraction and amplification, and a cgh array . In all, 41 were from the coq10 group and 74 from the placebo group . The rate of aneuploidy in the coq10 embryos was 46.5% and in the placebo group was 62.8% . Two of the transfers in each group were done five days after the retrieval whereas all the rest were done on day 3 . Three patients did not have a transfer for the lack of any normal embryos (one in the coq10 group and two in the placebo group). The clinical pregnancy rate was 33.3 and 26.7%, and live - birth rate was 25 and 26.7% for the coq10 and placebo groups, respectively . The decline in reproductive outcome associated with late maternal age is becoming a significant public health concern because of the current tendency of women to delay their childbirth . Thus far, there is no intervention apart from donor eggs capable of negating the adverse effects related to age . There is plenty of data supporting a role for the rate of metabolism and mitochondrial function with aging and longevity.22,23 small animals with a high rate of metabolism show an accelerated rate of aging in general as well as an accelerated reproductive senescence.2426 this allows for the use of mice as a model to study ovarian aging as well as potential interventions.27 in our animal model, we were able to show that prolonged exposure of elderly mice to coq10 was able to improve oocyte mitochondrial function, reduce the rate of decline of ovarian reserve, and restore reproductive outcome to a level comparable to young mice . Wilding and colleagues associated the age - related decline in oocyte mitochondrial function to a dysfunction of the spindle apparatus and an abnormal segregation of chromosome leading to aneuploidy.9 in the present study, we treated patients undergoing ivf icsi with either coq10 or placebo for two months before and up to oocyte retrieval, and compared the rate of oocyte aneuploidy between the two groups . Owing to the premature termination of the study for safety concerns, we were not able to reach the target number of patients defined by a power analysis . The results show a lower rate of aneuploidy in the coq10 group although the results did not achieve statistical significance . One simple explanation for the lack of significant differences between the coq10 and the placebo groups is the small sample size . Based on a power calculation, the study had to include a minimum of 27 patients in each arm . However, because of the premature termination of the study, the coq10 group had only one - third and the control group half of the target number . Another possible explanation for the difference in outcome between our human and animal study is the relative length of time in which coq10 was given . In the mouse study, the animals were treated for a quarter of their life span before being super - ovulated compared to only two months in humans . Coq10 is an essential part of the cellular energy production machinery; however, it is also a potent cellular antioxidant . There are data showing that cells severely deficient in coq10 show a reduced atp concentration that can be corrected following one week of incubation with coq10.28 aging was associated with differing levels of coq10 deficiency both in rats and humans . Even in the very old age group, in most tissues the decline of coq10 concentrations was not below 50% of peak concentration.29 ovary is an organ that shows preferential absorption of exogenous coq10.30 we can, therefore, make the assumption that treatment with coq10 for two months may have been sufficient to restore normal energy production in the ovary . Recently, several publications studying the origin of the age - related increase in aneuploidy have pinpointed it to the second meiotic division, and in the vast majority of cases, it appears to be the result of premature separation of sister chromatids.31,32 this phenomenon is mostly attributed to a dysfunction of two nuclear proteins cohesin complex and shugoshins which in addition to crossover chiasmata maintain sister chromatid cohesion during meiosis . In addition, cohesin complexes and shugoshins have been shown to lose function after prolonged exposure to ros.33 tarin et al have shown that the administration of the antioxidants vitamins e and c was able to correct some of the age - related decline in reproduction in mice.34 quinzii et al used a model of gradual inhibition of coq10 production creating different levels of coq10 deficiency.29 they were able to show that at the level of coq10 typically seen in the elderly, the effect observed in mitochondrial function is not a decline in atp production, but rather an increase in ros production . In reviewing these data, we conclude that the administration of coq10 for two months, although sufficient to correct a potential lack of atp in the oocyte, might not be enough to prevent the effects of prolonged exposure to ros on the meiotic apparatus . More research is needed to study the effects of a longer duration of intake of coq10 on female reproductive function in women of various ages.
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Therapeutic drug monitoring (tdm) of antiepileptic drugs (aeds) is a common practice and is usually done to optimize their dosing regimens, efficacy and in investigating therapeutic failure or patient compliance and avoid toxicity . As seizures occur irregularly, sometimes with long gaps in between the episodes, long - term administration and therefore observation of aed therapy becomes necessary to assess the patient compliance and its clinical benefit . Some aeds produce adverse effects which are difficult to distinguish from underlying neurologic disease and finally, there are no laboratory tests or diagnostic procedures available that can easily assess the clinical efficacy of the aeds . Patient demographics such as age, gender, and bodyweight play a key role in the pharmacokinetic variability of these drugs . Measuring serum levels is also indicated in patients with conditions such as hepatic or renal impairment and pregnancy which alter their pharmacokinetic characteristics so as to maintain there effective drug levels . On the other hand, it is also a fact that if pharmacokinetic parameters of a drug are consistent and predictable, then dosing of that drug can often be done without the need for tdm which is usually seen with newer aeds . Levetiracetam (lev) is one of the newer aed which became available in the year 2000 . Although there exists a linear relationship between the dose and the serum levels of lev, some studies have suggested that serum levels of lev can get affected by a number of parameters . Reference ranges (levels) for the newer aeds like lev have been difficult to establish . Ideally, tdm guides physicians toward that serum concentration of the drug which would optimize the seizure control, while avoiding or at least minimizing their toxic effects . However, it has been seen that a particular individual may show a good clinical response at aed concentrations even outside the reference range for that drug . Based on this understanding, serum levels of lev were measured and correlated with the patient's demographics and clinical response, based on the assumption that the clinical effects for certain diseases correlate better with drug concentrations than with the dose given . While tdm is an established method to individualize the dosage of aeds since many years, its impact on clinical course in patients with epilepsy has rarely been assessed especially with new aeds in a systematic manner . Patients suffering from epilepsy (any seizure semiology) and started on lev were enrolled for the study, after obtaining their written informed consent, from the outpatient department of neurology of a tertiary care medical college and hospital of north india from july 1, 2014 to august 31, 2015, after approval from institutional ethics committee . As this was a pilot study, 31 patients above 18 years of age and started on lev were selected but only 29 completed the study [figure 1]. Patients who were pregnant, having a history of renal insufficiency, liver disease, or any other comorbid condition were excluded . Flow chart of patient recruitment patient demographics such as age, gender, height, and bodyweight along with clinical characteristics such as seizure frequency, detailed history of aeds intake both past and present and other concomitant treatment received were recorded . The blood samples of these patients were collected for estimation of serum levels of lev (after dose stabilization), creatinine, alanine transaminase (alt), aspartate transaminase (ast), and albumin . Serum levels of lev in these patients were measured using shimadzu's high - performance liquid chromatography (hplc) system (lc-2010 aht / cht). C18 column was used as stationary phase and triethylamine buffer (10 ml triethylamine in 1000 ml water) adjusted to ph 6.5 0.2 by phosphoric acid and acetonitrile in the ratio of 85:15 respectively was used as mobile phase . Flow rate of the mobile phase was 0.8 ml / min with a run time of 8 min . Unknown patient serum samples were run against the standard calibration curve of lev prepared which was linear with a correlation coefficient (r) of 0.999 . The limit of detection and quantification of this assay method were 0.01 and 0.03 g / ml, respectively . The determined serum levels of lev were correlated with age, gender, bodyweight, dose used, formulation, compliance, therapeutic effect, suspected toxicity, suspected drug interactions, and adverse drug reactions (adrs) in the patients . Figure 1 shows the recruitment of patients in a flow chart . Among the 29 patients who were included in the study, 15 were males and 14 were females . Serum levels of creatinine, alt, ast, and albumin were within normal ranges for these patients . Other than lev, patients were also on clobazam, valproic acid (va), oxcarbazepine, carbamazepine, phenytoin, phenobarbitone, lamotrigine, and topiramate in various combinations . Demographic characteristics of the study subjects the mean dose given to the patients in the study was 26.45 10.76 mg / kg / day with a range of 7.5750.00 mg / kg / day whereas the median total daily dose of lev given was 1500 mg (range 5004000 mg). The median serum level of lev obtained at different time intervals from the drug intake and blood sampling was 17.8 g / ml with a range of 0.4102.2 g / ml as shown in table 2 . Figure 2 illustrates the range of serum lev concentration at total dose given per day in milligrams . It shows that there is no increase in the serum lev levels with an increase in doses . Figure 3 shows the distribution of serum lev levels of patients versus time gap since blood sample collection after the last dose in semi - logarithmic scale . Table 3 shows the serum lev levels with respect to age, gender, bodyweight and formulation . For making comparisons, patients with similar doses and blood sampling time since the last dose were taken . For comparing the effects of age and bodyweight, patients were divided into below and above 40 years of age and 60 kg bodyweight, respectively . Correlation (r) between serum lev concentration with dose administered (mg / kg / day), age and bodyweight all patients were on lev tablets except one each (elderly at 73 and 65 years age) on syrup and intravenous injection . Dosage and serum concentration of levetiracetam in the study subjects total levetiracetam dose versus serum concentration levetiracetam concentration versus time comparison of serum levetiracetam levels with respect to age, gender, bodyweight, and formulation all patients were compliant as they had detectable levels of lev in their serum but whether they were strictly adherent to drug therapy cannot be commented upon as the levels show wide variations . 22 out of 29 (75.86%) patients showed> 50% reduction in seizure frequency / week out of which 18 were completely seizure free . Two showed 50% reduction, three showed no change, and one showed 50% increase in seizure frequency / week as shown in figure 4 . The patient with increased frequency of seizure episodes was suffering from absence seizures and was diagnosed with bilateral temporal lobe due to epilepsy . One patient suffered from frequent oral twitching due to post hypoxic brain injury, but there was no record of the frequency of these twitchings . Table 4 shows serum lev concentrations with and without aed comedication whereas table 5 shows a comparison of serum levels of two patients each, on similar doses of lev while on concomitant use of other aeds . Although taken at different time intervals since drug intake, the serum levels of lev were 7.8% higher in patients on concomitant drug va and 56.8% lower in patients on concomitant inducer drugs (carbamazepine, phenobarbitone, phenytoin) as compared to those in patients on concomitant neutral drugs (clobazam, lamotrigine, oxcarbazepine, topiramate) or lev monotherapy . Serum lev levels of these patients were within or below the normal trough levels of serum lev and had no correlation with these adrs . Change in seizure frequency versus serum levetiracetam concentrations serum concentration of levetiracetam with and without antiepileptic drug comedication in the study subjects antiepileptic drug interactions among the study subjects in this study, the serum lev levels (nontrough) showed a negligible negative correlation with the dose of lev given per kilogram (kg) bodyweight . (n = 69) obtained a negligible positive correlation (r = 0.29) while may et al . (n = 297) obtained a moderately positive correlation (r = 0.67) between trough serum lev concentration and dose per kg bodyweight . Although there exists a linear relationship between the dose and the trough serum levels of lev over a dose range of 5005000 mg, some studies have however suggested that serum levels of lev can get affected by a number of parameters such as age, bodyweight, comedication, hepatic, or renal insufficiency . Furthermore, the lev concentrations depend on the time interval from the last dose taken, and because of short half - life (68 h), large fluctuations are expected during the day; as a result, nontrough levels of lev could not be well correlated with the dose . Serum levels of lev showed negligible positive correlation with the age of the patients as also seen by mathew et al . This is in conjunction with a study conducted by may et al . According to which older patients need a lower lev dose per bodyweight than young adults to achieve comparable lev levels . It has been concluded that the older adults have lower clearance (cl) than younger adults and therefore require a mean 40% lower dose of lev to achieve the same serum level . Age affect the apparent clearance of lev to the largest extent as shown by a 40% reduction in the elderly compared with the adults . This difference in the serum concentration of lev can be due to the difference in the clearance rate of the drug from the kidney with age . Three studies done on the correlation of serum lev levels with gender came out with a conclusion that gender had no significant effect on the serum levels, and/or clearance of lev but another study found that females had 12% higher lev exposure than males . In the present study, only two patients (one male and one female) could be compared as blood samples for them were collected at the same time gap (3.5 h), where the female patient showed a higher concentration of lev as compared to the male patient . This could be due to the differences in the bodyweight between the two genders (males are heavier), but due to limited number of comparable subjects, no significant conclusions could be drawn as in regard to the effect of gender on serum lev levels . Radtke in his study (n = 391) showed that any differences in the pharmacokinetic parameters (if present) are likely related to the differences in bodyweight of both the genders and show no differences when normalized for bodyweight . According to pigeolet et al . (n = 524) bodyweight has a statistically significant effect on apparent plasma clearance of lev and its volume of distribution . Decreasing bodyweight from 70 kg to 40 kg increased lev exposure by 16% . In this study also, the serum levels were found to be higher in 60 kg patients as compared to> 60 kg patients, and the correlation between serum lev levels and bodyweight was negligibly negative against that seen by mathew et al . (r = 0.12) in a study (n = 24), it was found that all pharmacokinetic parameters of both oral and liquid formulations of lev were bioequivalent and therefore no adjustment in dosage is necessary if a patient is switched from one formulation to another . In this study, all patients except two were on oral tablets . The only patient on syrup was elderly (73 years) and showed a higher concentration of lev which could be due to the age . On the other hand, other patient (age 65 years) on intravenous injection had a high concentration which could also be due to the age or a higher dose [table 3]. Therefore, no clear conclusion could be drawn of the effect of formulations on the serum concentration of lev . All patients showed compliance to drug therapy as they had detectable levels of lev in their serum, but they may or may not be strictly adherent to the dosage schedule as these levels were measured at different time gaps from the drug intake and blood sample collection and vary widely with a range of 0.4102.2 g / ml . The values on the lower side of this range are well below the established lower trough range of lev (before the scheduled next dose) in the western studies (12 g / ml) while patients still showing improvement in their seizure frequency . There was no observed relationship of the clinical response seen in our patients with the serum concentration of lev . This was similar to what was seen in the study conducted by sheinberg et al ., (n = 50) where 95% of the patients had more than a 50% reduction of seizure frequency; only one patient had increased seizure frequency, and two had no change in seizure frequency after start of lev . Coadministration of enzyme inducer aeds reduced the serum concentration of lev as compared to the coadministration of neutral aeds whereas coadministration of va increased the lev concentration [table 4]. Decrease in serum levels of lev occurs due to the increase in its clearance (cl / f) by concomitant use of enzyme inducer aeds which have an inducible effect on the enzymes (plasma esterases, hydrolases) involved in the lev metabolism . On the other hand, fat gain related to increased food consumption increase in serum levels of lev with va occurs due to change in body water composition as lev dissolves in water . The volume of distribution (v / f) of lev is decreased by va . (n = 629) showed that drowsiness and psychiatric / behavioral effects were the most common adverse effects associated with lev use . Sheinberg et al . Found that there was no relationship between serum concentrations of lev and adverse events . In this study also, we found that the most common adrs were somnolence and aggressiveness and that they could not be correlated with the serum concentrations of lev . There is no cut off limit of serum levels of lev in the literature above which toxicity appears . Only three patients showed serum levels of lev above upper limit of trough therapeutic range (46 g / ml). As these levels have been obtained at 3 h (87.1 g / ml), 3.5 h (73.6 g / ml), and 14 h (102.2 g / ml) gap of drug intake and blood sample collection, they may not suggest toxic levels even otherwise . Despite being a pilot study with a small sample size, the present study has shown that the serum lev levels are affected by the age, bodyweight and use of comedications and can also predict patient compliance . A study with a larger sample size in the future although the purpose of the present project was to study the ranges of serum lev concentrations irrespective of the time gap between the last dose taken and the blood sample collection, a future study with blood samples collected at the same time after the drug intake, preferably the trough samples just before the next morning dose of lev, should be preferably done as lev shows variations in its serum levels throughout the day . Better comparisons can also be made if patients are enrolled with same total doses instead of different doses . Despite being a pilot study with a small sample size, the present study has shown that the serum lev levels are affected by the age, bodyweight and use of comedications and can also predict patient compliance . A study with a larger sample size in the future although the purpose of the present project was to study the ranges of serum lev concentrations irrespective of the time gap between the last dose taken and the blood sample collection, a future study with blood samples collected at the same time after the drug intake, preferably the trough samples just before the next morning dose of lev, should be preferably done as lev shows variations in its serum levels throughout the day . Better comparisons can also be made if patients are enrolled with same total doses instead of different doses . Aed therapy is prescribed quite commonly at all levels of patient care, i.e., both rural and urban clinical practice . Using newer aeds is quite costly but nevertheless necessary as regard to their advantages over the older well - established counterparts . However, guided administration of these drugs is quite imperative for the purpose of rational prescribing and long - term patient care . This study was done to find the lev concentrations irrespective of the timing of last doses for the purpose of population pharmacokinetic studies . The study emphasizes that the tdm of newer aeds like lev should be done in routine clinical practice so as to adjust the dose according to age, bodyweight, and comedication and prevent its adverse effects . More studies on tdm of newer aeds is required on a large number of indian patients to develop a pooled database for generating the pharmacokinetic data ranges for our population . Newer aeds should be prescribed based on the tdm data available so as to prevent the irrational practice of medicine, complications of adrs, toxicity, and various drug - drug interactions.
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Intracellular signaling pathways transmit signals of various extracellular stimuli to their cytosolic and nuclear targets in order to induce biological responses, such as proliferation, differentiation, cell death and migration . When needed, the signals are transmitted from the cytoplasm to the nucleus via translocation of one or more components of each of the signaling pathways involved . Thus, after stimulation, a large number of signaling proteins are rapidly translocated to the nucleus to induce and regulate many nuclear processes . However, despite the importance of stimulated nuclear signaling, the mechanisms by which these components reach the nucleus upon stimulation have been elucidated only for a few signaling pathways . Classic nuclear shuttling is mediated by an importin- complex that binds to cargoes containing a nuclear localization signal (nls), consisting of mono- or bi - partite clusters of basic amino acids [1 - 3]. This importin- complex often acts as a housekeeping mechanism that shuttles most nuclear proteins immediately to the nucleus after their translation . The relocalization of cargoes is followed by the dissociation of the proteins from the importins upon binding to rangtp, which exports the importins back to the cytoplasm, while the cargo remains in the nucleus . However, only a limited number of signaling proteins, such as nfb and erk5 (extracellular signal - regulated kinase 5) [8 - 10], use this machinery for their stimulated nuclear shuttle . Aside from this canonical mechanism, importin- or similar karyopherins, termed -like importins, operate by binding to non - canonical nlss to mediate translocation without the assistance of importin-. The mechanism by which these components operate is much less understood and is currently under investigation . Recently, several -like importins have been implicated in the stimulated nuclear translocation of signaling proteins . Here, we propose that this group of importins might be responsible for the swift nuclear shuttling of many proteins following various stimuli . The rapid and robust activation of erk1/2 allows the phosphorylation and modulation of the activity of more than 300 proteins, which are localized either in the cytoplasm or the nucleus [13 - 15]. These substrates are important for the induction and regulation of cellular processes, including proliferation, differentiation, and migration amongst others [16 - 19]. The sub - cellular localization of erk1/2 plays an important role in its regulation and physiological functioning . Interestingly, it was shown that the nuclear accumulation of erk1/2 is important primarily for the induction of proliferation, while other erk - dependent processes are mostly regulated by cytosolic molecules . Erk1/2 localization, as well as the mechanisms that govern it, has been elucidated over the past decades . In resting cells, all components of the erk1/2 cascade are localized primarily in the cytoplasm due to their interaction with different anchoring proteins [25 - 28] (see figure 1). Upon stimulation, mek1/2 phosphorylates erk1/2 in their tey motif, thereby inducing a conformational change resulting in the activation of erk1/2 and detachment from their anchors . This detachment exposes erk1/2 to an additional phosphorylation on two ser residues (an sps motif) within a nine amino acid sequence, termed nuclear translocation signal (nts). This phosphorylation can be mediated by both stimulated and constitutively active protein kinases, including protein kinase ck2 and auto - phosphorylation by active erk1/2 . The phosphorylation of the sps motif allows it to bind importin-7, which escorts erk1/2 molecules to the nuclear pores, inducing nuclear sliding . Once in the nucleus, rangtp dissociates importin-7 from erk1/2, and consequently, induces their nuclear accumulation . It was also shown that erk1/2 may interact directly with the nuclear pores, and it is possible that these direct interactions are able to facilitate the erk1/2 translocation . In addition, this process may be regulated by calcium, as a reduction in intracellular calcium concentrations was shown to induce faster nuclear shuttling . The following steps are illustrated: (a) binding of erk1/2 with anchor proteins in resting cells; (b) stimulation is followed by phosphorylation of the tey motif of erk1/2 by mek1/2, and detachment of erk1/2 from their anchors; (c) phosphorylation of erk1/2 on its sps motif by ckii . (d) binding of phosphorylated erk1/2 to importin-7 and nuclear sliding through the nups; (e) dissociation of erk1/2 from importin-7 by rangtp, and nuclear accumulation of erk1/2 . For more details, see text . Interestingly, these results in mammals were consistent with findings in drosophila, where dim-7 (the ortholog of importin-7) was identified as the carrier of d - erk to the nucleus . Once in the nucleus, erk plays a critical role in the development of eyes and wings in drosophila . Moreover, while comparing the mechanism of nuclear translocation of components of the erk cascade with other proteins, we established that the nts might act as a specific stimulus - induced and importin-7-dependent nuclear translocation signal for some signaling proteins lacking an nls . However, since many signaling proteins contain neither nls nor nts, it is possible that other ill - defined -like importins participate in the stimulated translocation, using various non - canonical nlss . Although importin- complexes mediate the nuclear shuttling of a large number of proteins, it is now clear that other karyopherins are required for the translocation of the full repertoire of nuclear proteins . Such karyopherins were initially discovered as nucleoporin - binding proteins, and their homology with importin- suggested a function in nuclear transport [39 - 43], which initiated their importin terminology (see table 1 for nomenclature). Subsequently, more dedicated studies identified at least 10 more -like importins in mammals that share a sequence motif related to the ran - binding site of importin-, and can shuttle to the nucleus under various conditions . The -like importins known today share low overall sequence identity (10 - 20%), and have 19 - 20 helical heat repeats arranged into super - helical or ring - like structure . Their molecular weights (90 - 150 kda), and isoelectric points (pi = 4.0 5.9) vary . These importins mediate the translocation of proteins into the nucleus under varying conditions, including stimulation . Here, we describe the possible involvement of the -like importins, as well as exportin-4, in the stimulated translocation of signaling proteins . Modified from: eldar and seger mol cell biol (2013), submitted . Among the -like importins with the highest number of identified cargoes is importin-7, which seems to utilize several mechanisms and distinct nlss to shuttle its distinct cargoes (table 2). In some cases, usually in non - stimulated cells, importin-7 acts in a complex with importin-, or in parallel to importin-2, importin-4, and importin-8 . Thus, importin-7 mediates the nuclear translocation of erk1/2 described above, as well as mek1 and smad3, by binding to the nts sequences of these cargoes . Moreover, it was reported that importin-7 is able to directly bind to a canonical nls sequence in the glucocorticoid receptor to escort it to the nucleus upon hormonal stimulation . In addition, importin-7 seems to shuttle other signaling proteins or transcription factors to the nucleus in an nts- and canonical nls - independent manner . Such molecules include the transcriptional regulators hif1-, c - jun, several smad proteins, sox-2, hiv-1, egr-1, and the oncogenic npm - alk . However, it remains unclear whether the importin-7-mediated translocation of all these proteins is affected by stimulation, or is active merely in resting cells . Although the information on other members of the family still lags behind that of importin-7, it seems that at least some of them play important roles in stimulated translocation as well . Accordingly, importin-8 was shown to induce the nuclear accumulation of ago2 and smad1/4 . C - jun was shown to be transported by importins 2, 5, 7, 9, and 13, that might be related, at least in part, to its stimulated nuclear accumulation . C - fos to the nucleus after translation or upon stimulation, and importin-4 escorts vitamin d receptor to the nucleus upon ligand stimulation . Interestingly, exportin-4, which participates mainly in nuclear export, has been shown to function as an importin for sox-2, in addition to importin-7 and importin-9 . This makes exportin-4 a distant relative of the -like importins (table 1), although it is not clear whether it participates in stimulated translocations as well . In general, -like importins are able to induce both stimulated and/or non - stimulated translocations, using at least 4 mechanisms: (i) monomeric, direct canonical nls - dependent cargo binding; (ii) monomeric, direct non - canonical nls - independent cargo binding; (iii) cargo binding in a complex with importin-; and (iv) cooperation with other -like importins (see more details in table 2). Thus, as a group, -like importins may play an important role in the stimulated translocation of signaling proteins and transcription factors . There is increasing evidence that the translocation of signaling proteins into the nucleus is much more tightly regulated than it was thought just a few years ago . Aside from the nls / importin- machinery, other mechanisms, such as passive diffusion, active transport of homodimers [64 - 66], direct binding to nuclear pore machinery [31,67 - 70], escort to the nucleus by other nls - containing proteins and indirect aid by the canonical machinery, were initially proposed for several signaling proteins . However, some of these findings were not properly verified, and later were either disputed [73 - 75], or found to be cell - type specific . Therefore, it is worthwhile to entertain the possibility that at least some of these alleged mechanisms are, in fact, part of the wider -like importin - dependent networks . In addition, since dysregulation of the signaling proteins described above is involved in diseases, such as cancer and autoimmunity, it would be interesting to study the potential therapeutic implications of inhibiting their nuclear translocation . Several attempts have been made to block canonical nls / importin- mediated nuclear translocation [77 - 80]. Since many proteins use this machinery to translocate to the nucleus, such inhibition might affect too many processes and may fail to develop into desired specific therapies . However, a more specific approach might be to target the non - canonical mechanism of translocation, which seems to act within a limited number of distinct proteins upon stimulation . In this direction this peptide is able to compete with natural substrates and is resistant to ran - mediated release in the nucleus, therefore specifically inhibiting this process . However, in order to develop strong inhibitors for a specific cargo/-like importin complex, we need to extract precise information on the structural interaction, as well as the regulation of import . We will then be able to explore this mechanism as a new layer of therapeutic intervention.
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Chronic itch, which is defined as itch lasting more than 6 weeks, is a prevalent problem that occurs in ~10% of the population (mollanazar et al ., 2015). Chronic itch conditions negatively affect quality of life, and yet there are no therapies that are both efficacious and selective for itch . The lack of effective treatment is partly attributable to a poor understanding of the mechanisms that underlie it . Although antihistamines are frequently prescribed as a treatment for itch, they are typically ineffective because most types of chronic itch are not histamine - mediated (mollanazar et al ., 2015). Unfortunately, while there are numerous mediators that can cause itch, the factors that are responsible in most circumstances of chronic itch are largely unknown . Human psychophysical studies have shown that application of serotonin into the skin causes itch (weisshaar et al ., 2004). In rodents, serotonin is a key component of mast cells, and it is a potent mediator of itch . However, until recently, the mechanisms through which serotonin causes itch have remained uncertain . Many pruritogens bind to metabotropic receptors on primary sensory neurons; however, these receptors must be coupled to ionotropic channels via intracellular signaling pathways in order to allow sufficient current influx to generate action potentials . Several groups have shown that the cation channels trpv1 and trpa1 are coupled to different pruritogen receptors and that they are critical for different forms of itch transmission (ross, 2011). More specifically, trpv1 is required for histaminergic itch, whereas trpa1 is required for several types of non - histaminergic itch, such as that induced by chloroquine, bam8 - 22, il-31, endothelin-1, thymic stromal lymphopoietin, and bile acids . Until recently, whether serotonin receptors were likewise coupled to trps remained unknown . Understanding serotonin - mediated itch has been complicated by the fact that there are numerous serotonin receptors that are expressed on primary afferents, as well as on immune mediators that could be involved in itch . It was previously hypothesized that the primary pathway through which serotonin causes itch is via stimulation of histamine release from mast cells . However, contrary to this idea, antihistamines failed to reduce serotonin - induced itch sensation in humans (hosogi et al ., 2006). A recent study has demonstrated that one way in which serotonin induces itch is via direct activation of 5-ht7 (encoded by htr7), which is expressed on subsets of primary sensory afferents (morita et al ., 2015). In this study, mice lacking either htr7 or trpa1 showed substantially reduced scratching behavior in response to an intradermal injection of a 5-ht7-selective agonist . Furthermore, htr7 and trpa1 knockout mice scratched considerably less in a model of atopic dermatitis . However, it seemed likely that this was only part of the serotonin - itch story, because the 5-ht2-selective agonist, -methyl-5ht, is a potent pruritogen in mice . As reported in this issue of jid, the study by carstens and colleagues (2015) provides further insight into the molecular players involved in serotonin - evoked itch by defining a trpv4-dependent pathway that is likely to be downstream of 5-ht2-mediated itch . The original goal of this study was to investigate a possible role for trpv4 in itch . Trpv4 is upregulated in the skin of individuals with certain itch conditions (moore et al ., 2013; yang et al ., 2015 interestingly, trpv4 knockout mice displayed a significant reduction in scratching behavior in response to serotonin, but not to histamine, chloroquine, or sligrl (akiyama et al . A trpv4 antagonist also reduced substantially the amount of serotonin - evoked scratching, supporting the idea that trpv4 is critical to serotonin signaling in normal mice . Importantly, the authors showed that the change in response to serotonin in the trpv4 knockout mice was specifically a decrease in serotonin - evoked itch behaviors, and not a change in serotonin - evoked pain behaviors . This study demonstrates that trpv4 is a key downstream component of serotonin - evoked itch (figure 1). In order to link serotonin to trpv4 and the activation of sensory neurons serotonin - mediated activation was dependent on trpv4, as a trpv4 antagonist reduced significantly the calcium response to the application of serotonin . In support of this finding, the authors demonstrated that the proportion of neurons that responded to serotonin was reduced significantly in trpv4 knockout mice . Interestingly, the proportion of neurons responding to other types of pruritogens did not change in mice lacking trpv4, indicating that trpv4 plays an important and specific role in responses to serotonin in primary sensory neurons . To identify the receptor through which serotonin acts, akiyama et al . The 5-ht2 antagonist, but not the 5-ht1 antagonist, reduced serotonin - evoked scratching . This finding raises the possibility that 5-ht2, acting via trpv4, is key mediator of serotonin - evoked itch . Thus, there appear to be at least two distinct pathways through which serotonin mediates itch: a trpa1-dependant pathway that mediates 5-ht7-mediated itch, as well as a trpv4-dependent pathway that likely mediates 5-ht2-mediated itch . What remains to be tested is whether these receptors are expressed on distinct or overlapping populations of primary sensory afferents.
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Parkinson's disease (pd) is the second most common neurodegenerative disorder after alzheimer's disease in the elderly . Clinically, pd is characterized by tremor, rigidity, slowness of movement, and postural imbalance . Although its etiology is not fully understood, pd is suggested to involve interactions between a susceptible genetic background and environmental factors . Vitamin d, which is now considered a hormone rather than a vitamin, is suggested to be an environmentally modifiable factor in the pathogenesis of pd recently . A long - term cohort study from finland suggested that low serum vitamin d is a predictive factor for high risk of pd . The vitamin d receptor (vdr) is the key mediator of vitamin d's functions . Upon binding to 1, 25-dihydroxy vitamin d3, the biologically active form of vitamin d, vdr is activated and interacts with vitamin d responsive elements in the promoters of vitamin d target genes to regulate their expression . Interestingly, vdr is most highly expressed in dopaminergic neurons of the substantia nigra and an earlier animal study with knockout of vdr gene resulted in rats with muscular and motor impairments . All these suggested a role of vdr in the pathogenesis of pd . Previous results showed that variations in the vdr gene influence receptor activation by altering the affinity of the receptor for vitamin d. recently, butler et al . Suggested that the vdr gene is a candidate gene for promoting pd and then a number of restriction fragment length polymorphisms (rflp) in this gene have been studied . Among the accumulating studies on the association between vdr gene and pd in recent years, polymorphisms of apai (rs7975232), bsmi (rs1544410), foki (rs10735810), and taqi (rs731236) have been most extensively investigated, but results to date are inconsistent . The discrepancies in findings may be attributed to small sample sizes, clinical heterogeneity, statistical power or a combination of these factors . To overcome the limitations of individual studies, we performed a meta - analysis to investigate the effects of these polymorphisms on pd . Relevant publications were identified by conducting a literature search in pubmed, embase, ebsco, china national knowledge infrastructure, weipu database, and wanfang database up to january 2015, with the following terms and combinations: vdr, calcitriol receptors, polymorphism, single nucleotide polymorphism, variant, pd . The references of the identified studies and reviews were additionally screened to determine other potential eligible studies . The inclusion criteria were as follows: (1) case control studies; (2) studies analyzing the association of apai, bsmi, foki, and taqi gene polymorphisms and pd risk; (3) those reporting sufficient genotype data to calculate the odds ratio (or) and corresponding 95% confidence interval (ci) and; (4) cases in the control group are publication - based or hospital - based which are matched for gender and age . Studies were excluded for the following reasons: (1) case reports or reviews; (2) family - based studies; (3) insufficient genotype data for calculation and; (4) genotype distribution of controls not in hardy - weinberg equilibrium (hwe), indicating the chance of bias during control selection or genotyping errors . In addition, we checked the polymerase chain reaction primer sequence in genebank (http://www.ncbi.nlm.nih.gov/genebank/index.html) to confirm whether the selected genes were the target genes . The quality of studies was independently assessed by the two reviewers using the newcastle - ottawa scale (nos) based on three aspects: selection, comparability, and exposure of cases and controls . Nos scores ranged from 0 to 9, and studies with a score equal to or higher than six were regarded as high quality . Two investigators reviewed the publications independently and extracted the relevant information from each eligible study based on criteria in a standard data form, including name of first author, year of publication, ethnicity of the study population, number of cases and controls, matching criteria, source of controls, allele and genotype frequency, genotyping method, and evidence of hwe in controls . Cochrane review manager version 5.3 (cochrane library, oxford, uk) was used to calculate available data from each study . The pooled or and 95% ci were calculated to measure the genetic association between vdr polymorphisms and risk of pd in four genetic models, including additive genetic model (m vs. w), codominant model (mm vs. ww and mw vs. ww), dominant model (mm + mw vs. ww) and recessive model (mm vs. mw + ww). Here, m represent wild - type and mutant type, respectively . The significance of or was determined with the z - test, in which p <0.05 was considered statistically significant . The degree of heterogeneity between studies was detected using the q - test and i - statistics . I values of 25%, 50%, and 75% were assigned as low, moderate, and high estimates, respectively . The fixed - effects model was adopted at pq> 0.05 or i <50% . Sensitivity analysis was conducted by omitting each study to assess the stability of the results . Publication bias was assessed using begger's funnel plots and egger's linear regression test by stata 12.0 software (stata coporation, tx, usa). Relevant publications were identified by conducting a literature search in pubmed, embase, ebsco, china national knowledge infrastructure, weipu database, and wanfang database up to january 2015, with the following terms and combinations: vdr, calcitriol receptors, polymorphism, single nucleotide polymorphism, variant, pd . The references of the identified studies and reviews were additionally screened to determine other potential eligible studies . The inclusion criteria were as follows: (1) case control studies; (2) studies analyzing the association of apai, bsmi, foki, and taqi gene polymorphisms and pd risk; (3) those reporting sufficient genotype data to calculate the odds ratio (or) and corresponding 95% confidence interval (ci) and; (4) cases in the control group are publication - based or hospital - based which are matched for gender and age . Studies were excluded for the following reasons: (1) case reports or reviews; (2) family - based studies; (3) insufficient genotype data for calculation and; (4) genotype distribution of controls not in hardy - weinberg equilibrium (hwe), indicating the chance of bias during control selection or genotyping errors . In addition, we checked the polymerase chain reaction primer sequence in genebank (http://www.ncbi.nlm.nih.gov/genebank/index.html) to confirm whether the selected genes were the target genes . The quality of studies was independently assessed by the two reviewers using the newcastle - ottawa scale (nos) based on three aspects: selection, comparability, and exposure of cases and controls . Nos scores ranged from 0 to 9, and studies with a score equal to or higher than six were regarded as high quality . Two investigators reviewed the publications independently and extracted the relevant information from each eligible study based on criteria in a standard data form, including name of first author, year of publication, ethnicity of the study population, number of cases and controls, matching criteria, source of controls, allele and genotype frequency, genotyping method, and evidence of hwe in controls . Cochrane review manager version 5.3 (cochrane library, oxford, uk) was used to calculate available data from each study . The pooled or and 95% ci were calculated to measure the genetic association between vdr polymorphisms and risk of pd in four genetic models, including additive genetic model (m vs. w), codominant model (mm vs. ww and mw vs. ww), dominant model (mm + mw vs. ww) and recessive model (mm vs. mw + ww). Here, m represent wild - type and mutant type, respectively . The significance of or was determined with the z - test, in which p <0.05 was considered statistically significant . The degree of heterogeneity between studies was detected using the q - test and i - statistics . I values of 25%, 50%, and 75% were assigned as low, moderate, and high estimates, respectively . The fixed - effects model was adopted at pq> 0.05 or i <50% . Sensitivity analysis was conducted by omitting each study to assess the stability of the results . Publication bias was assessed using begger's funnel plots and egger's linear regression test by stata 12.0 software (stata coporation, tx, usa). In total, 60 studies relevant to search keywords were identified, from which 30 repeated publications, 10 reviews and 8 irrelevant polymorphism studies were excluded, leaving 12 studies . After a full - text review, three of these studies were excluded, as they were not case - control analyses, and two were excluded owing to insufficient genotype data [figure 1]. Ultimately, seven case - control studies involving 2034 pd patients and 2432 healthy individuals met our inclusion criteria ., four focused on apai, four on bsmi, two on foki, and four on taqi gene polymorphisms . All genotype distributions in the controls in this meta - analysis did not deviate from hwe [table 1]. Characteristics of the eligible studies snp: single nucleotide polymorphism; pb: population - based; hb: hospital - based; hwe: hardy - weinberg equilibrium; nos: newcastle - ottawa scale; pcr - rflp: polymerase chain reaction - restriction fragment length polymorphism . The results of the assessment of correlations between vdr polymorphisms and pd are shown in tables 2 and 3 . Meta - analysis was performed for a combined asian and caucasian population and by each individual ethnic category . In overall population, no evidence of significant association between apai (g / t), bsmi (g / a) or taqi (t / c) polymorphisms and pd was observed under all genetic models [table 2]. For foki (c / t) polymorphism, we found it was significantly associated with pd risk under the additive, codominant model and dominant model (c vs. t: or = 1.41, 95% ci: 1.141.75, p = 0.001; cc vs. tt: or = 2.45, 95% ci: 1.523.93, p = 0.0002; ct vs. tt: or = 2.21, 95% ci: 1.383.52, p = 0.0009, cc vs. ct+tt: or = 2.32, 95% ci: 1.493.61, p = 0.0002). Meta - analysis of the association between vdr gene polymorphism and pd or: odds ratio; ci: confidence interval; vdr: vitamin d receptor; pd: parkinson s disease; snp: single nucleotide polymorphism . Subgroup analysis of the association between the vdr gene polymorphism and pd or: odds ratio; ci: confidence interval; vdr: vitamin d receptor; pd: parkinson s disease; snp: single nucleotide polymorphism . Consistently, ethnicity - specific analysis revealed no association between apai, bsmi or taqi polymorphisms and pd risk while a significant association between foki (c / t) polymorphisms and pd was observed in both asian and caucasian population [table 3]. A sensitivity analysis was additionally conducted in which one study at a time was removed . The results showed no significant alterations in pooled or and 95% ci values, indicating that our analysis is statistically robust . No between - study heterogeneity was found in analyses of the apai, bsmi or taqi polymorphisms in the combined population . Visual inspection of funnel plots did not find any evidence of funnel plot asymmetry [figure 2]. Statistical result of egger's test till did not show publication bias among studies [table 2]. Funnel plots for vitamin d receptor gene polymorphisms and parkinson's disease ([a] t vs. g of apai; [b] a vs. g of bsmi; [c] c vs. t of foki; [d] c vs. t of taqi). In total, 60 studies relevant to search keywords were identified, from which 30 repeated publications, 10 reviews and 8 irrelevant polymorphism studies were excluded, leaving 12 studies . After a full - text review, three of these studies were excluded, as they were not case - control analyses, and two were excluded owing to insufficient genotype data [figure 1]. Ultimately, seven case - control studies involving 2034 pd patients and 2432 healthy individuals met our inclusion criteria ., four focused on apai, four on bsmi, two on foki, and four on taqi gene polymorphisms . All genotype distributions in the controls in this meta - analysis did not deviate from hwe [table 1]. Characteristics of the eligible studies snp: single nucleotide polymorphism; pb: population - based; hb: hospital - based; hwe: hardy - weinberg equilibrium; nos: newcastle - ottawa scale; pcr - rflp: polymerase chain reaction - restriction fragment length polymorphism . The results of the assessment of correlations between vdr polymorphisms and pd are shown in tables 2 and 3 . Meta - analysis was performed for a combined asian and caucasian population and by each individual ethnic category . In overall population, no evidence of significant association between apai (g / t), bsmi (g / a) or taqi (t / c) polymorphisms and pd was observed under all genetic models [table 2]. For foki (c / t) polymorphism, we found it was significantly associated with pd risk under the additive, codominant model and dominant model (c vs. t: or = 1.41, 95% ci: 1.141.75, p = 0.001; cc vs. tt: or = 2.45, 95% ci: 1.523.93, p = 0.0002; ct vs. tt: or = 2.21, 95% ci: 1.383.52, p = 0.0009, cc vs. ct+tt: or = 2.32, 95% ci: 1.493.61, p = 0.0002). Meta - analysis of the association between vdr gene polymorphism and pd or: odds ratio; ci: confidence interval; vdr: vitamin d receptor; pd: parkinson s disease; snp: single nucleotide polymorphism . Subgroup analysis of the association between the vdr gene polymorphism and pd or: odds ratio; ci: confidence interval; vdr: vitamin d receptor; pd: parkinson s disease; snp: single nucleotide polymorphism . Consistently, ethnicity - specific analysis revealed no association between apai, bsmi or taqi polymorphisms and pd risk while a significant association between foki (c / t) polymorphisms and pd was observed in both asian and caucasian population [table 3]. A sensitivity analysis was additionally conducted in which one study at a time was removed . The results showed no significant alterations in pooled or and 95% ci values, indicating that our analysis is statistically robust . No between - study heterogeneity was found in analyses of the apai, bsmi or taqi polymorphisms in the combined population . Visual inspection of funnel plots did not find any evidence of funnel plot asymmetry [figure 2]. Statistical result of egger's test till did not show publication bias among studies [table 2]. Funnel plots for vitamin d receptor gene polymorphisms and parkinson's disease ([a] t vs. g of apai; [b] a vs. g of bsmi; [c] c vs. t of foki; [d] c vs. t of taqi). Although the multifactorial nature of pd is well - recognized, genetic factors are considered the significant determinants . Numerous genes have been studied in connection with pd, and the vdr gene has been one of the research hotspots recently . It is suggested that genetic polymorphisms in the vdr gene alter the affinity of the receptor for vitamin d, which may be an environmentally modifiable factor in the pathogenesis of pd . Given the potential association between vdr and pd, vdr polymorphisms affecting its expression have been analyzed to determine their potential effects on pd . The human vdr gene contains eight exons and seven introns spanning> 75 kb dna on chromosome 12q12 . To date, over 60 polymorphisms have been identified for vdr genes . These subtle dna sequence variations, which often occur in the population, have a modest but real biological effect . Among these, apai, bsmi, foki, and taqi polymorphisms have been most intensively investigated in relation to pd as well as other neurodegenerative diseases . However, controversial results have been obtained . To establish the association of vdr apai, bsmi, foki, and taqi polymorphisms with risk of pd, we performed a meta - analysis of seven studies (including 2034 pd cases and 2432 controls). We failed to detect any association between apal, bsml, taql polymorphisms and pd susceptibility in all four genetic models . A previous study suggested strong linkage disequilibrium for bsmi, apai, and taqi rflps, which may explain the high degree of consistency between these three polymorphisms and their link to pd susceptibility . These three polymorphisms are located at the 3end of the vdr gene neighboring the 3utr region, which do not result in changes in the predicted amino acid sequence of the vdr . However, one thing we should pay attention to is that the serum vitamin d level, which most of the included studies did not measure, may influence the effect size of a vdr polymorphism . In a study analyzing association between vdr polymorphisms and incidence of type i diabetes mellitus, the or of the risk allele increasing with higher vitamin d levels in the studied population . A recent report that the positive association between vdr genetic variants and multiple sclerosis was found in studies conducted in regions at lower latitudes but rather than higher latitudes . Therefore, it is important to include measurement of serum vitamin d levels in future studies to better characterize the genetic effects of vdr polymorphisms in pd . Our data suggest that foki (c / t) polymorphism is significantly associated with pd and the c allele may increase the risk of pd . The foki polymorphism is located in exon 2 at the 5 coding region of the gene . Previous studies by gross et al . Suggested that vdr encoded by the c allele is three amino acids shorter than that encoded by the t allele . However, since there are only two studies on the foki (c / t) gene polymorphism have been documented, one in an asian and the other in a caucasian population . Although the results from both investigations were consistent, data should be carefully interpreted because of the relatively small sample sizes . Additional studies with larger sample sizes are necessary to verify the role of foki polymorphisms in pd . First, the number of cases and controls involved in the meta - analysis were limited, which could increase the probability of false positives or false negatives . Second, although publication bias among studies were not detected, and the quality of our studies was good (nos6), the possibility of bias could not be eliminated because of the relatively small sample size . Third, factors such as gene - gene or gene - environment interactions may influence the gene - disease factor . However, the lack of individual data from the included studies limited further evaluation of these potential interactions . Data from our meta - analysis suggest that the apai, bsmi, and taqi gene polymorphisms are not significantly associated with pd while the foki (c / t) polymorphism may increase pd risk . Given the limitations mentioned above, further studies with more specific common information and larger sample sizes are warranted . Furthermore, additional investigations are required to determine the biological mechanisms underlying the observed associations and their potential relationship with pd.
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Reading a journal article from the primary scientific literature can be a daunting task for anyone especially for k to 12 students and teachers . The barriers that inhibit people who are not professional scientists from being able to read a scientific article include: finding and accessing an article of interest; comprehending the field - specific jargon (8); and understanding how to navigate the structure of the article to locate the desired information . We at the journal of emerging investigators (jei, www.emerginginvestigators.org) aim to lower these barriers for middle and high school students and their mentors . (for a deeper discussion of these issues and how jei addresses them, please see the accompanying perspectives article in this issue (3).) Increasing student engagement with the scientific literature is important because it is the most unbiased and direct way to learn scientific information . Moreover, it encourages students to think critically about the process of how scientific facts are discovered and teaches them how to apply the scientific method to their own questions . We also believe that making students comfortable with primary literature articles will break down the damaging stereotype that science is impenetrable and only for experts . Eliminating this commonly held but incorrect belief is one of the critical first steps in empowering scientific competency in all citizens . The internet is a powerful tool to help students hurdle the first two barriers described above . Articles now exist that can be easily accessed and searched for articles of interest, which can be downloaded in full (table 1). However, once an article is in hand and jargon is defined, primary literature can still be very difficult to navigate for anyone unfamiliar with its foreign, formal structure . We sought to create a fun, easy learning tool to help familiarize students and their teachers, parents, and other mentors with the structure of a scientific article . Our main learning objective was for the student to realize that science writing is formulaic that specific information is found in predictable and defined locations within an article and that, with an understanding of the formula, anyone can comfortably navigate any journal article and accurately predict what to expect to find in each section . Therefore, we designed the journal article scavenger hunt to require the user to find and identify a series of commonplace features of a primary research article (fig . 1). This activity quickly highlights the similarities found across all journal articles, and it can be scaled up or down in difficulty to the level of the user . At jei, we have had success using the scavenger hunt with students of all ages, from elementary school children to lay adult - learners, and in a variety of settings, from the classroom and lab to outreach booths at fairs . While many pedagogical approaches already exist to help students learn from primary literature (e.g., create (5), figure facts (7), and others summarized in 7 and 6), most are geared toward using articles to teach college students scientific content and/or processes and require significant investments of classroom time . Jei s journal article scavenger hunt complements these approaches by providing a novel focus on teaching the structure of a scientific article, rather than its content, in a manner that is accessible to school - aged children and can be accomplished in as little as five minutes . Each website contains thousands of scientific articles that are all open - access and can be downloaded for free for use in the classroom, lab, or for personal interest . To find an article of interest note: all articles from the journal of emerging investigators are written by students, whereas all other sources contain articles written by professional researchers . This generic template for the journal article scavenger hunt can be modified to suit the needs of the instructor . The questions can be adjusted to be more or less challenging, and/or more or less specific to the content of the journal article of choice . 1), writing utensils, and one or more primary research articles . For outreach events that engage the public, such as booths at science festivals, supplemental materials can be helpful, including visual aids that relate to the articles (e.g., sealed moldy agar plates for articles on microbes) and inexpensive incentives (e.g., neon bouncy balls for kids who complete the scavenger hunt). However, these items do not directly relate to the scavenger hunt itself and are not necessary to complete it . Primary research articles to be used with the scavenger hunt can be pre - selected by the instructor and distributed with the activity, or they can be found as part of the student s preparation for the activity (see table 1 for where to find articles). For example, when working with older teenagers and college students, we choose articles like the relationship between sleep and wake habits and academic performance in medical students: a cross - sectional study (2). This research asks whether late bedtimes and daytime sleepiness correlate with poor grades in school a question that this age group easily relates to and enjoys . For younger or broader audiences we recommend using articles published in the journal of emerging investigators, where the research is designed, performed, and written by student authors in grades 6 to 12 . These articles tend to have simpler methodologies and intuitive subjects such as the effect of music on heart rate (1) or the isolation of microbes from common household surfaces (4). While being approachable, jei articles are still peer - reviewed, primary research articles and so they conform to the standard of professional literature in terms of structure and layout and serve as excellent introductions to the science writing format . The activity itself is straightforward: each student receives one copy of the scavenger hunt for each article (fig . The worksheet is comprised of fill - in - the - blank questions that alert the reader s attention to common features in all primary research articles, such as the authors, the main research question being asked, the publication dates, and the main conclusion of the work . The instructions state every scientific primary research article contains the following items can you find them all? The student must then scan the research article to find the items and write in the correct answers . The student should not statically read the article from beginning to end; rather, the activity should be done quickly by scanning for the pertinent information, with most students completing it within five minutes . For more mature and independent students, the activity can be performed individually but it is often more fun and interactive when done in small groups . In classroom settings, format, where the pairs share their explanations with the entire class to generate discussion . We have found that the best way for students to grasp the structural similarities that all articles share is to have students perform the scavenger hunt iteratively with multiple, disparate articles . The students quickly recognize the pattern of where to find the desired information and can then more quickly find the same information in a new article . To emphasize this point and to build excitement, we make it a race: we use stopwatches to time how quickly a student can accurately complete the scavenger hunt for one article, and then see if s / he can beat that time when finding the same information in a new article . We have used it as a pre - lab assignment or as homework with high - school students and college freshmen to guide their reading of a primary research article . We have also had great success with the scavenger hunt as an outreach activity at science fairs, where we use prizes and races to incentivize engagement from children as young as elementary school - age . We find that students of all ages find comfort in the predictability of where to find specific information within the foreign format of the article, and we see their scavenger hunt completion times decrease and their confidence grow in real time as they quickly succeed in finding correct answers . Similarly, we have used the scavenger hunt in professional development seminars for middle and high school science teachers to de - mystify the structure of the journal article and to encourage teachers inclusion of primary literature in their classrooms . Teachers evaluations of the scavenger hunt were universally positive, with one teacher writing that she now feels able to introduce the structure of science writing to my [6 grade] students for the first time . To more formally measure the efficacy and impact of the journal article scavenger hunt, jei is currently in the process of developing summative assessments of its publication and outreach efforts (see our accompanying article published in this themed issue (3) for further information). Not only is the scavenger hunt activity beneficial for various audiences and settings, the hunt itself is adaptable to the instructor s needs . The questions within the scavenger hunt can be modified by the instructor to add variety to the assignment each year or to accommodate different learning styles . The content of the scavenger hunt can also be tailored to specific articles of interest to meet the instructor s learning goals . For example, when we at jei use the scavenger hunt to teach about the peer - review process, we include a bonus question that draws the reader s attention to the difference between the submission date and the publication date . An example of a modified scavenger hunt used with college freshmen can be found in appendix 1 . 1), writing utensils, and one or more primary research articles . For outreach events that engage the public, such as booths at science festivals, supplemental materials can be helpful, including visual aids that relate to the articles (e.g., sealed moldy agar plates for articles on microbes) and inexpensive incentives (e.g., neon bouncy balls for kids who complete the scavenger hunt). However, these items do not directly relate to the scavenger hunt itself and are not necessary to complete it . Primary research articles to be used with the scavenger hunt can be pre - selected by the instructor and distributed with the activity, or they can be found as part of the student s preparation for the activity (see table 1 for where to find articles). For example, when working with older teenagers and college students, we choose articles like the relationship between sleep and wake habits and academic performance in medical students: a cross - sectional study (2). This research asks whether late bedtimes and daytime sleepiness correlate with poor grades in school a question that this age group easily relates to and enjoys . For younger or broader audiences we recommend using articles published in the journal of emerging investigators, where the research is designed, performed, and written by student authors in grades 6 to 12 . These articles tend to have simpler methodologies and intuitive subjects such as the effect of music on heart rate (1) or the isolation of microbes from common household surfaces (4). While being approachable, jei articles are still peer - reviewed, primary research articles and so they conform to the standard of professional literature in terms of structure and layout and serve as excellent introductions to the science writing format . The activity itself is straightforward: each student receives one copy of the scavenger hunt for each article (fig . The worksheet is comprised of fill - in - the - blank questions that alert the reader s attention to common features in all primary research articles, such as the authors, the main research question being asked, the publication dates, and the main conclusion of the work . The instructions state every scientific primary research article contains the following items can you find them all? The student must then scan the research article to find the items and write in the correct answers . The student should not statically read the article from beginning to end; rather, the activity should be done quickly by scanning for the pertinent information, with most students completing it within five minutes . For more mature and independent students, the activity can be performed individually but it is often more fun and interactive when done in small groups . In classroom settings, format, where the pairs share their explanations with the entire class to generate discussion . We have found that the best way for students to grasp the structural similarities that all articles share is to have students perform the scavenger hunt iteratively with multiple, disparate articles . The students quickly recognize the pattern of where to find the desired information and can then more quickly find the same information in a new article . To emphasize this point and to build excitement, we make it a race: we use stopwatches to time how quickly a student can accurately complete the scavenger hunt for one article, and then see if s / he can beat that time when finding the same information in a new article . We have used it as a pre - lab assignment or as homework with high - school students and college freshmen to guide their reading of a primary research article . We have also had great success with the scavenger hunt as an outreach activity at science fairs, where we use prizes and races to incentivize engagement from children as young as elementary school - age . We find that students of all ages find comfort in the predictability of where to find specific information within the foreign format of the article, and we see their scavenger hunt completion times decrease and their confidence grow in real time as they quickly succeed in finding correct answers . Similarly, we have used the scavenger hunt in professional development seminars for middle and high school science teachers to de - mystify the structure of the journal article and to encourage teachers inclusion of primary literature in their classrooms . Teachers evaluations of the scavenger hunt were universally positive, with one teacher writing that she now feels able to introduce the structure of science writing to my [6 grade] students for the first time . To more formally measure the efficacy and impact of the journal article scavenger hunt, jei is currently in the process of developing summative assessments of its publication and outreach efforts (see our accompanying article published in this themed issue (3) for further information). Not only is the scavenger hunt activity beneficial for various audiences and settings, the hunt itself is adaptable to the instructor s needs . The questions within the scavenger hunt can be modified by the instructor to add variety to the assignment each year or to accommodate different learning styles . The content of the scavenger hunt can also be tailored to specific articles of interest to meet the instructor s learning goals . For example, when we at jei use the scavenger hunt to teach about the peer - review process, we include a bonus question that draws the reader s attention to the difference between the submission date and the publication date . An example of a modified scavenger hunt used with college freshmen can be found in appendix 1 . The journal article scavenger hunt is a fun, adaptable activity that quickly familiarizes the user with the structure of a primary scientific research article . Over many years of using this activity, we have received positive feedback from students of all ages, from elementary school students to lay adult - learners as well as science teachers themselves . By making the unknown seem predictable and approachable, the scavenger hunt helps a variety of audiences feel more comfortable with science and more confident in their ability to engage directly with the scientific literature.
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Despite the advances made in medical science, diabetes mellitus continues to be a life - threatening disease . Insulin and hypoglycemic agents have increased the life span of diabetic patients, but the associated complications continue to be the cause of morbidity and mortality . Beta cell function is gradually lost, requiring increasing doses of oral hypoglycemic drugs and/or requiring insulin . In search of new remedies for diabetes, the plant argyrolobium roseum came under authors investigation on the basis of information that a person residing in a distant hilly area had treated some cases of diabetes by 2-week treatment with a plant growing in that area . On visit to the said place, the concerned person was contacted, the plant locally known as fly jari was collected, botanically identified, and a sample was preserved in the herbarium of the institute labeled as asc-29 . On review of literature, it was found that though the plant had been mentioned in some texts,[13] no clinical use was attributed to this plant . However, a fraction of argyrolobium roseum has now been reported for its insulin secretagogue activity . When tested on normal fasting rats, the plant powder and its alcohol and acetone extracted fractions exhibited hypoglycemic activity . Since merely the hypoglycemic effect of the plant could not be accounted for recovery of the patient from diabetes by 2-week treatment, this led us to think that the plant possibly possessed beta - cell neogenesis activity . To explore this activity, an experimental model with parameters indicative of the status of the beta cells argyrolobium roseum (family leguminosae; sub - family fabaceae) is a small ground level growing plant . The whole plant was plucked from ground level in the flowering month of march, shade dried, powdered, and kept in the refrigerator before it was processed . The powdered plant was percolated four times with methyl alcohol (90%), vacuums dried, coded as ar, and was employed for detailed study . From this extract, these were evaluated for hypoglycemic activity and compared with tolbutamide and glipizide as the reference standards . The test material was given orally in the form of a fine homogenized aqueous suspension prepared with 1% (w / v) gum acacia . Review of literature of the hypoglycemic activity possessing medicinal plants and their active constituents revealed that some cyclitols including d - pinitol having insulin - like activity are present in some natural sources like pine needles, chickpeas, alfalfa, soya beans, and other legumes and in bougainvillea spectabilis. [57] as the plant under study belongs to leguminosae family, it was deemed possible that active constituent present could be a cyclitol; study was planned accordingly to isolate it . Ar was washed with petroleum ether followed by wash with chloroform; five washes with both were given . The aqueous part was collected and passed through a column (60 cm height 2.5 cm diameter) filled with ion exchange resin first basic, i.e., amberlite (ir 400) followed by acidic (ir 120). The resins retain reducing sugars, pigments, and ions, while cyclitols including pinitol elute out . The eluted solution was vacuum - dried, dissolved in methanol, and crystallization was allowed to occur at 4c followed by filtration of mother liquor and subsequently, the crystalline material collected was air dried . The crystallized substance was subjected to determination of its melting point and high - performance liquid chromatography (hplc) (shimadzu, chennai, india). The separation was carried out on rezex rso - oligosaccharide ag+4% column of size 200 10 mm . Hplc - grade water was used as mobile phase and pumped at a flow rate of 0.3 ml / min . Confirmation of presence of pinitol was done by matching the retention time of the peak in the sample . Wistar rats, male and female, weighing 200 10 g fed on standard pellet diet, water ad libitum, and maintained at 24 to 28c room temperature with 12-hour day and night cycle were used . The hyperglycemic rats employed were streptozotocin (stz) treated, 40 mg / kg i.p . Blood was withdrawn by retro - orbital puncture using a fine sterile capillary tube for glucose estimation, before and 3 hours after test drug treatment . Vehicle (1% w / v gum acacia aqueous suspension) in equivalent amount as per body weight served as the control . Glipizide in a dose of 0.5 mg / kg p.o . Served as the reference standard . Serum was separated within 30 minutes and samples were assayed for glucose estimation by the trinder's method . Five groups of fasting rats, with six rats in each group, were employed . Each group of rat was for the specific interval of time, i.e.,, 1, 2, 3, and 5 hours . The blood sugar level of rats in each group before and after treatment with ar for each interval of time was determined . This method is different from the conventional methods of using the same group of animals repeatedly for different intervals of time, as the acute stress associated with repeated loss of blood and infliction of injury due to repeated withdrawal of blood from the same animal is likely to distort the blood sugar level of the animals and thereby interfering with the effect of the test drug . Stz is reported to induce diabetes with features similar to that associated with uncontrolled diabetes mellitus in human subjects . Stz has been employed at different doses in different species of animals,[1012] and reported to damage beta cell by generation of free radicals[1316] and il - ib . One of the recognized models for beta cell neogenesis is the neonatal rat injected with stz at the time of birth . The evidence for beta cell neogenesis in this model was based partly on cytological and partly on pharmacological investigations . The former involved immunochemical and stereological morphometric methods we had neither the facilities nor the expertise to carry out the cytological investigation; so, it was compelling on our part to devise a reliable and relevant model for generating pharmacological evidence on the neogenesis of beta cell . It was deemed more relevant to include adult rats as in them stz caused pathological pattern that resembled type 2 diabetes . The dose of stz reported for induction of diabetes is 50 to 60 mg / kg by intraperitoneal (ip) or intravenous (iv) routes, with death of animals within a week . It was decided to use such a dose of stz that caused mortality over a period of 3 to 4 weeks (sublethal dose), permitting at least two intervening weeks for the treatment of animals with test drug, before the animal dies . For this, different doses of stz were tried and a dose of 40 mg / kg / ip was found to be appropriate . The use of this dose and route had been reported earlier also . On day 0, the animals showing blood sugar level in normal range were treated with the said dose of stz . On day seven, their blood sugar was determined and accordingly, the animals were distributed into respective groups so that the mean blood sugar value of each group was close to each other . Treatment with test drug or controls the drug treatment was discontinued after day 21, but the animals were monitored for their beta cell status for the next three weeks . At the end of every week, blood sugar level, mortality, and body weight were recorded as indicator of functional status of the beta cell . On day 42, the response of the survived animals to the hypoglycemic effect of ar or glipizide was tested as this reflected the status of the beta cells . Results were expressed as mean sem and p value <0.05 was considered statistically significant . Argyrolobium roseum (family leguminosae; sub - family fabaceae) is a small ground level growing plant . The whole plant was plucked from ground level in the flowering month of march, shade dried, powdered, and kept in the refrigerator before it was processed . The powdered plant was percolated four times with methyl alcohol (90%), vacuums dried, coded as ar, and was employed for detailed study . From this extract, these were evaluated for hypoglycemic activity and compared with tolbutamide and glipizide as the reference standards . The test material was given orally in the form of a fine homogenized aqueous suspension prepared with 1% (w / v) gum acacia . Review of literature of the hypoglycemic activity possessing medicinal plants and their active constituents revealed that some cyclitols including d - pinitol having insulin - like activity are present in some natural sources like pine needles, chickpeas, alfalfa, soya beans, and other legumes and in bougainvillea spectabilis. [57] as the plant under study belongs to leguminosae family, it was deemed possible that active constituent present could be a cyclitol; study was planned accordingly to isolate it . Ar was washed with petroleum ether followed by wash with chloroform; five washes with both were given . The aqueous part was collected and passed through a column (60 cm height 2.5 cm diameter) filled with ion exchange resin first basic, i.e., amberlite (ir 400) followed by acidic (ir 120). The resins retain reducing sugars, pigments, and ions, while cyclitols including pinitol elute out . The eluted solution was vacuum - dried, dissolved in methanol, and crystallization was allowed to occur at 4c followed by filtration of mother liquor and subsequently, the crystalline material collected was air dried . The crystallized substance was subjected to determination of its melting point and high - performance liquid chromatography (hplc) (shimadzu, chennai, india). The separation was carried out on rezex rso - oligosaccharide ag+4% column of size 200 10 mm . Hplc - grade water was used as mobile phase and pumped at a flow rate of 0.3 ml / min . Confirmation of presence of pinitol was done by matching the retention time of the peak in the sample . Wistar rats, male and female, weighing 200 10 g fed on standard pellet diet, water ad libitum, and maintained at 24 to 28c room temperature with 12-hour day and night cycle were used . The hyperglycemic rats employed were streptozotocin (stz) treated, 40 mg / kg i.p . Dissolved in 0.01 m citrate buffer, 10 days after stz treatment . Blood was withdrawn by retro - orbital puncture using a fine sterile capillary tube for glucose estimation, before and 3 hours after test drug treatment . Vehicle (1% w / v gum acacia aqueous suspension) in equivalent amount as per body weight served as the control . Glipizide in a dose of 0.5 mg / kg p.o . Served as the reference standard . Serum was separated within 30 minutes and samples were assayed for glucose estimation by the trinder's method . Five groups of fasting rats, with six rats in each group, were employed . Each group of rat was for the specific interval of time, i.e.,, 1, 2, 3, and 5 hours . The blood sugar level of rats in each group before and after treatment with ar for each interval of time was determined . This method is different from the conventional methods of using the same group of animals repeatedly for different intervals of time, as the acute stress associated with repeated loss of blood and infliction of injury due to repeated withdrawal of blood from the same animal is likely to distort the blood sugar level of the animals and thereby interfering with the effect of the test drug . Stz is reported to induce diabetes with features similar to that associated with uncontrolled diabetes mellitus in human subjects . Stz has been employed at different doses in different species of animals,[1012] and reported to damage beta cell by generation of free radicals[1316] and il - ib . One of the recognized models for beta cell neogenesis is the neonatal rat injected with stz at the time of birth . The evidence for beta cell neogenesis in this model was based partly on cytological and partly on pharmacological investigations . We had neither the facilities nor the expertise to carry out the cytological investigation; so, it was compelling on our part to devise a reliable and relevant model for generating pharmacological evidence on the neogenesis of beta cell . It was deemed more relevant to include adult rats as in them stz caused pathological pattern that resembled type 2 diabetes . The dose of stz reported for induction of diabetes is 50 to 60 mg / kg by intraperitoneal (ip) or intravenous (iv) routes, with death of animals within a week . It was decided to use such a dose of stz that caused mortality over a period of 3 to 4 weeks (sublethal dose), permitting at least two intervening weeks for the treatment of animals with test drug, before the animal dies . For this, different doses of stz were tried and a dose of 40 mg / kg / ip was found to be appropriate . The use of this dose and route had been reported earlier also . On day 0, the animals showing blood sugar level in normal range were treated with the said dose of stz . On day seven, their blood sugar was determined and accordingly, the animals were distributed into respective groups so that the mean blood sugar value of each group was close to each other . Treatment with test drug or controls was started from day seven and continued till day 21 . The drug treatment was discontinued after day 21, but the animals were monitored for their beta cell status for the next three weeks . At the end of every week, blood sugar level, mortality, and body weight were recorded as indicator of functional status of the beta cell . On day 42, the response of the survived animals to the hypoglycemic effect of ar or glipizide was tested as this reflected the status of the beta cells . Results were expressed as mean sem and p value <0.05 was considered statistically significant . Fall in blood sugar levels were 24, 29, 21, and 18 mg / dl with plant powder, its alcoholic extract, acetone extracted fraction, and glipizide, respectively, in normal fasting rats [table 1]. The aqueous and alkaloid fractions did not show this activity (data not shown). When hypoglycemic activity of ar was tested on diabetic rats, ar showed significant fall of blood sugar in stz - treated hyperglycemic rats . This hypoglycemic activity continued from half - hour of ar treatment till 5th hour (last recording) [table 2]. Hypoglycemic activity of argyrolobium roseum and its fractions on fasting rats onset and duration of hypoglycemic effect of alcoholic extract of ar on fasting rats no adverse effect or mortality was observed on any animal treated with ar or plant powder in the dose of 200 and 2 500 mg / kg po, respectively . In control group(s), blood sugar continued to rise and remain at high level, the body weight continued to fall, and there was 100% mortality by day 28 in male group and 40% in female group by day 42 . In the ar - treated group, blood sugar after initial rise and body weight after initial reduction returned back to normal and there was no mortality . On day 42 of study, ar - treated group animals responded to hypoglycemic effect of ar equivalent to that recorded in normal rats, indicating thereby that the functional status of beta cells was as good as that of normal animals [tables 3 and 4]. Beta cells neogenesis by ar in streptozotocin - treated male rats beta cells neogenesis by ar in streptozotocin - treated female rats to check if hypoglycemic effect of ar was contributing in any way to its beta cell neogenesis activity, glipizide as a hypoglycemic agent was used as a positive control . In glipizide - treated group, decrease in blood glucose levels was less marked during the course of treatment as compared with ar group and it increased more after the treatment was stopped . Body weight continued to decrease as compared with its rise in ar - treated group . On day 42, glipizide tested on lone surviving rat showed hypoglycemic effect [table 5]. Beta cells neogenesis by ar vs glipizide in streptozotocin treated male rats tests were repeated with half the dose of ar used as compared with earlier tests, i.e., 100 mg / kg compared with 200 mg / kg employed in earlier test . Although the dose of 200 mg restored the blood sugar and body weight back to near normal without any mortality and the animals responded positively to the hypoglycemic effect of the ar on day 42 of study [tables 3 to 5], the dose of 100 mg of ar did not produce the effects to the same extent, though the effect was better than the control group [table 6]. Beta cells neogenesis with low dose of ar in streptozotocin - treated female rats hplc of the ar showed a peak retention time of 34.033 minutes, which is the same as that of d - pinitol (34.017). Fall in blood sugar levels were 24, 29, 21, and 18 mg / dl with plant powder, its alcoholic extract, acetone extracted fraction, and glipizide, respectively, in normal fasting rats [table 1]. The aqueous and alkaloid fractions did not show this activity (data not shown). When hypoglycemic activity of ar was tested on diabetic rats, ar showed significant fall of blood sugar in stz - treated hyperglycemic rats . This hypoglycemic activity continued from half - hour of ar treatment till 5th hour (last recording) [table 2]. Hypoglycemic activity of argyrolobium roseum and its fractions on fasting rats onset and duration of hypoglycemic effect of alcoholic extract of ar on fasting rats no adverse effect or mortality was observed on any animal treated with ar or plant powder in the dose of 200 and 2 500 mg / kg po, respectively . In control group(s), blood sugar continued to rise and remain at high level, the body weight continued to fall, and there was 100% mortality by day 28 in male group and 40% in female group by day 42 . In the ar - treated group, blood sugar after initial rise and body weight after initial reduction returned back to normal and there was no mortality . On day 42 of study, ar - treated group animals responded to hypoglycemic effect of ar equivalent to that recorded in normal rats, indicating thereby that the functional status of beta cells was as good as that of normal animals [tables 3 and 4]. Beta cells neogenesis by ar in streptozotocin - treated male rats beta cells neogenesis by ar in streptozotocin - treated female rats to check if hypoglycemic effect of ar was contributing in any way to its beta cell neogenesis activity, glipizide as a hypoglycemic agent was used as a positive control . In glipizide - treated group, decrease in blood glucose levels was less marked during the course of treatment as compared with ar group and it increased more after the treatment was stopped . Body weight continued to decrease as compared with its rise in ar - treated group . On day 42, glipizide tested on lone surviving rat showed hypoglycemic effect [table 5]. Beta cells neogenesis by ar vs glipizide in streptozotocin treated male rats tests were repeated with half the dose of ar used as compared with earlier tests, i.e., 100 mg / kg compared with 200 mg / kg employed in earlier test . Although the dose of 200 mg restored the blood sugar and body weight back to near normal without any mortality and the animals responded positively to the hypoglycemic effect of the ar on day 42 of study [tables 3 to 5], the dose of 100 mg of ar did not produce the effects to the same extent, though the effect was better than the control group [table 6]. Hplc of the ar showed a peak retention time of 34.033 minutes, which is the same as that of d - pinitol (34.017). D - pinitol (3-o - methyl chiro - inositol) is an inositol, a key component of the insulin - related phosphoglycans released from cell membrane on the binding of insulin with its receptor and likely to be participating in the release of phosphoglycans and thereby enhance the activity of insulin or overcome the insulin resistance . This explains for insulin - like effects observed with d - pinitol to improve glycemic control in a number of experimental and clinical studies reported. [72231] d - pinitol is reported to be present in pine needles, chickpeas, alfalfa, soya beans, and other legumes and in bougainvillea spectabilis used in traditional medicine for conditions associated with diabetes . The model employed to explore beta cell neogenesis activity is based on causing apoptosis of beta cell with stz . It was decided to use such a dose of stz that led to hyperglycemia, weight loss, and mortality but not before 2 to 3 weeks, the time period which was required for a test drug to act and show its beta cell neogenesis activity, if possessed . Accordingly, dose of 40 mg / kg i.p . As such, one week time was allowed for diabetes to be established, i.e., allow time for the beta cell to undergo apoptosis before the treatment was started, then continued for 2 weeks for the drug to act, observe in next 3 weeks without drug treatment for the recovery from diabetes, i.e., neogenesis of beta cells, and then determine the status of beta cells at the end, i.e., on day 42 by testing the response of a hypoglycemic agent, as has been done in this study and ar - treated animals which were found as recovered, i.e., beta cells regenerated as compared with controls . Thus, the pharmacological evidence recorded by way of restoration of blood sugar and body weight to near normal and absence of mortality as compared with controls and response of ar - treated animals to a hypoglycemic agent at the end of 6 weeks are indicative of beta cell neogenesis . The experimental model developed is all the more relevant since diabetes is first allowed to develop in adult rats as is the case with type 2 diabetes appearing in adult age . The recognized model of beta cell neogenesis in stz - treated neonatal rats has a drawback that damage to beta cells by stz in new born is followed by a rapid remission from neonatal diabetes starting from day 3 to 5 after birth . Adult rats with stz exhibit decreased beta cell mass and a chronic pathological pattern that presents functional similarities to type 2 diabetes . Secondly, the model developed can be put to use by those who do not have the facilities for carrying out cytological studies involving immunochemical and morphometric methods . The pancreatic beta cell responsible for maintenance of body's glucose level within a narrow range, their number, and functioning can be said to be dynamic, i.e., undergoing both replication and apoptosis in a balanced way . One determinant in the development of diabetes is inadequate mass of beta cells, either absolute in type 1 diabetes or relative in type 2 diabetes . A better understanding of regeneration and factors responsible for stimulating regeneration and replication can lead to new therapeutic strategies for the treatment of diabetes . If beta cell neogenesis by ar is further confirmed by cytological evidence, it will provide a new dimension to the therapeutic intervention of diabetes mellitus . Neogenesis from duct epithelium is the most currently described and best documented process of differentiation of precursor cells into beta cells . This process contributes not only to beta cell mass expansion during fetal and neonatal life, but it is also involved in the maintenance of beta cell mass in adults and further a number of factors controlling the differentiation of precursor cells have been identified. [3436] recently, the incretin hormone glucagon - like peptide (glp-1) and its long - acting analogue exendin-4, known to enhance glucose - stimulated insulin release and glucose disposal in peripheral tissues, have been reported to stimulate the proliferation of ins-1 cells in vitro, and increase beta cell mass in adult rodents in vivo. [3840] it is likely that d - pinitol present in ar is exerting both hypoglycemic and beta cell neogenesis by augmenting the release of glp-1, as glp-1 has been reported to mediate its action by promoting dna synthesis, activation of phosphotidylinostol 3-kinase and by increasing transcription factor . Follow - up study for recording cytological evidence on beta cell neogenesis by ar or its active constituents is warranted . It is also hoped that the experimental model developed for evaluating the beta cell neogenic activity of a test material is going to prove useful as a screening model for the said activity.
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Nursing students are constantly under various stressors such as academic performance, isolation from the group, change in environment, change in food type, irregular diet, clinical challenges, economic problems, future prospects, problems with the value system of current society, self - expectations, and expectations from family and peers etc1,2,3,4,5,6 . If these stressors are not managed properly, they can have negative effects on the physical and psychological health of nursing students1,2,3 . Furthermore, these problems can cause academic failure and/or limited growth as a valuable employee2, 4, 7 . Therefore, it is necessary to put in place various activities and behaviors to prevent and reduce life stress in nursing students4, 6,7 . All these stressors have adverse effects on the autonomic nervous system, pineal and hypothalamic - pituitary - adrenal axis that interrupt the healthy operation of the homeostatic mechanisms of the body8 . This has damaging effects on various parameters of the body, and one of the common parameters is blood glucose2 . It is observed that stress increases both short - term and long - term blood glucose levels, and if it increases one or both of them beyond the normal limits, stress can cause persistent high blood glucose levels, resulting in diabetes mellitus2 . Yoga, which integrates various postures, breathing exercises, and meditation, is beneficial in reducing stress . The mechanisms by which yoga influences well - being are likely complex biochemical and physiological mechanisms consisting of reduced sympathetic nervous tone, activation of antagonistic neuromuscular systems, and stimulation of the limbic system9 . That is to say, breathing exercises during yoga practice reduce sympathetic tone, increases parasympathetic activity, and help to reduce stress in yoga practictioners10 . Meditation also brings about a hypometabolic state and reduces stress induced by sympathetic overactivity11 . Also, yogic exercise has a direct influence on pancreatic secretion by rejuvenation of the pancreatic cells through alternate abdominal contractions and relaxation12, 13, and yogic exercise reduces blood glucose levels due to muscular exercise and relaxation12, 13 . The dynamic stretching of the body during yoga asanas is postulated to rejuvenate pancreatic cells, increase insulin secretion, and hence correct the impaired insulin secretion in chronic diabetes14 . Ultimately, yoga practice has been proven to affect mental balance of an individual by allying apprehension and stress and bringing about hormonal balance and feelings of well - being2 . In previous studies, researchers have indicated that yoga intervention played an effective role in decreasing stress and improving general well - being in medical students2, 15 . In spite of its known positive effects on physical and psychological variables, no studies have been conducted with yogic programs to manage nursing students life stress . Hence, the purpose of this randomized controlled trial study was to evaluate the effect of yogic exercises on life stress and biochemical markers of stress such as blood glucose levels in nursing students . The subjects of this study were 27 healthy female undergraduate nursing students aged 2023 years with no medical diseases, no history of drug abuse, and no experience with yoga exercise . All participants received detailed information on the purpose and usefulness of this study and were provided a written consent form . After the participants signed the informed consent form, they were randomly assigned by a random permuted block design using a random number table, to 1 of 2 groups, either the yoga exercise (n=15) or control group (n=15). Twenty - seven subjects completed the entire study, 12 in the yoga exercise group and 15 in the control group . Three subjects dropped out of the study before completing the posttest due to personal reasons . Demographic information, life stress scale, and postprandial blood glucose levels were measured as pretest data . The life stress scale and postprandial blood glucose levels were also measured as posttest data . This instrument consisted of 50 items scored using a 4-point likert scale (0=never, 3=very often) measuring sources of life stress in 8 subscales - economy, lover, faculty, family, the future, values, grades, and friends . A higher score indicates a higher level of perceived stress . In this study, the internal consistency (cronbach s alpha) of the life stress scale was 0.93 . After completing life stress scale surveys, measurements were taken from the participants both before commencement of the study and also after the yogic intervention was over . Postprandial blood glucose levels were measured with a digital glucometer (accu - chek . Sr . Yogic exercises were conducted for about one hour, once a week for 12 weeks . Cyclic form yoga exercises were chosen because of the availability of scientific studies on this type of yoga17, 18 . Also yoga nidra, which means psychic sleep, is a specific yogic relaxation and meditation practice19 . The cyclic yoga performed in this study consisted of the following practices (table 1table 1.yogic exercise processstep 1: surya namaskara (20 min/10 cycles)step 2: shavasana (5 min)step 3: yoga nidra (35 min/1 cycle)(postures and breathing exercise)(relaxation)(meditation and relaxation)1 . Pranamasana (prayer pose)). Under the guidance and supervision of the yoga expert, after practicing 10 cycles of surya namaskara for 20 minutes, the subjects performed the shavasana pose for 5 minutes of relaxation . Finally, the subjects performed yoga nidra . It has several steps, such as resolve, rotation of consciousness, awareness of the breath, feeling and sensation, visualization, and ending the practice with resolve . A t test was used to test for the homogeneity of demographic and clinical characteristics between the exercise and control group . The level of statistical significance was considered to be a probability value of less than 0.05 . No significant differences in age were found between the yoga group and the control group (table 2table 2.homogeneity test for age, life stress, and blood glucose level between the experimental and control groupvariablesexperimental groupmean sdcontrol groupmean sdage (years)21.0 0.221.0 0.3life stress (score)1.6 0.31.6 0.3economy1.6 0.51.7 0.5lover1.3 0.41.4 0.4faculty1.2 0.81.3 0.8family1.3 0.41.2 0.4future2.0 0.41.9 0.3value1.8 0.61.9 0.5grades2.4 0.42.2 0.5friend1.3 0.41.1 0.3ppg (mg / dl)90.0 11.589.6 9.9ppg: postprandial blood glucose, sd: standard deviation). There were no significant differences in the pre - intervention life stress scale score or pre - intervention postprandial blood glucose level between the two groups (table 2). Ppg: postprandial blood glucose, sd: standard deviation the life stress score significantly differed between the 2 groups (yoga group vs. control group, p<0.001) and over time (pretest vs. posttest, p<0.001). For the yoga group, the mean post - intervention life stress score (mean sd, 0.77 0.49) was significantly decreased compared with the mean pre - intervention life stress score (mean sd, 1.6 0.3). Of the life stress subscales, the life stress subscale scores of economy, lover, family, the future, values, grades, and friends were decreased significantly in the yoga group after the yogic exercises, respectively (table 3table 3.effects of yogic exercises on life stress and postprandial blood glucose levelgrouppretestposttestdifference (posttest - pretest)mean sdmean sdmean sdlife stress (score)yoga1.6 0.30.7 0.4***0.8 0.6***control1.6 0.31.7 0.20.1 0.3economyyoga1.6 0.50.6 0.5***1.0 0.6***control1.7 0.51.9 0.50.1 0.5loveryoga1.2 0.40.5 0.5**0.7 0.7**control1.4 0.41.5 0.60.1 0.7facultyyoga1.1 0.70.1 0.2***1.0 0.7control1.2 0.80.6 0.70.6 1.1familyyoga1.2 0.30.5 0.5**0.7 0.6***control1.2 0.41.4 0.40.1 0.4futureyoga2.0 0.31.1 0.7**0.8 0.8***control1.8 0.32.1 0.30.2 0.4valuesyoga1.8 0.50.7 0.6***1.0 0.7***control1.9 0.52.2 0.30.2 0.6gradesyoga2.4 0.41.6 0.7*0.7 0.8***control2.2 0.52.6 0.30.3 0.5friendsyoga1.2 0.30.6 0.5**0.6 0.6***control1.1 0.31.2 0.30.1 0.3ppg (mg / dl)yoga90.0 11.481.3 8.7**8.7 7.5***control89.6 9.9103.4 8.2**13.8 13.7*p<0.05; * * p<0.01; * * * p<0.001; ppg: postprandial blood glucose level, sd: standard deviation). * p<0.05; * * p<0.01; * * * p<0.001; ppg: postprandial blood glucose level, sd: standard deviation the postprandial blood glucose level significantly differed between the 2 groups (yoga group vs. control group, p<0.001) and over time (pretest vs. posttest, p<0.05). For the yoga group, the mean post - intervention postprandial blood glucose level (mean sd, 81.3 8.7) was significantly decreased compared with the mean pre - intervention postprandial blood glucose level (mean sd, 90.0 11.4). For the control group, the mean post - intervention postprandial blood glucose level (mean sd, 103.4 8.2) was significantly increased compared with the mean pre - intervention postprandial blood glucose level (mean sd, 89.6 9.9) (table 3). As shown in the study results, after 12 weeks of yogic exercises, life stress scale scores were significantly decreased compared with those before starting yoga practice . Additionally, nursing students in the yoga exercise group had a significant decrease in their stress levels over this 12-week period, while those in the control group had an increase in their stress levels . These findings are similar to the findings of simard and henry, who found that perceived stress scale levels significantly decreased from baseline to mid - term, an improvement that persisted to the end of the intervention15 . They assessed the impact of a short yoga intervention on female medical students health for a 16-week period . They also pointed out that a yoga intervention may be effective in decreasing stress and improving general well - being in medical students . Besides, the findings of beets & mitchell indicated that despite short yoga program exposure, acute changes in mental health indicators such as stress were observed20 . Similarly, the findings of yoshihara et al . Suggested that yoga can improve mental health status and has implications for the prevention of psychosomatic symptoms in healthy women17 . In addition, sharma systematically reviewed 17 articles related to yoga for stress management8 . As a result, they reported that of the 17 studies, 12 demonstrated positive changes in psychological or physiological outcomes related to stress . Like the previous studies, the present study also supports the paradigm that yoga exercise can have beneficial effects in reducing psychological problems such as stress in nursing students . Of the life stress this result is inconsistent with the concept that academic factors are a greater perceived cause of stress in nursing students1, 5, 6 . Over the past 2 decades, many forms of intervention have been suggested to help nursing students with their stress . Most recently, one researcher recommended biofeedback, humor, peer instructors, mentors, and mindfulness training interventions for stress centered around academic challenges in nursing students2, 5, 6, 21,22,23 . However, no recent studies have been conducted with a new generation of yoga exercises to help reduce nursing students stress . Ultimately, these findings suggest that the nursing students in the yoga exercise group were able to control the same level of stress over the 12-week period even though they experienced more stressors and demands from their academic conditions . It is very important for nurse educators to help nursing students manage their stress in order to prevent additional problems . In the present study, fasting blood sugar levels could not be checked because of the time of the yoga class as the participants personal situations . After practicing yoga for 12 weeks, postprandial blood glucose levels were significantly decreased in the yoga group . This finding is similar to that of shende et al ., who found that both fasting and postprandial blood glucose levels were significantly improved in the yoga group, whereas there was no significant improvement in the postprandial blood glucose levels in their control group not practicing yoga pranayama4 . They assessed the effect of pranayama on the blood glucose level of 60 medical students and found that a short - term intervention of yogic exercise helps in reducing the stress in medical students, which is reflected by improvement in both fasting and postprandial blood glucose levels . They also emphasized that the beneficial effects of yoga on fasting and postprandial blood glucose levels are well documented . As stated, most of these studies have been done on patients with known diseases . However, there is very little data available on the effect of yoga exercise composed of surya namaskara (which means sun salutations) and yoga nidra (which means psychic sleep) on the blood glucose levels in healthy subjects . The findings of the present study also support those of jensen, stevens, and kenny, who stated that yoga nidra is related to psycho - neuro - physiological parameters24 . Numerous studies have shown yoga to have an immediate downregulating effect on both the pineal and hypothalamic - pituitary - adrenal axis responses to stress12,13,14,15 . The performance of asana led to increased sensitivity of the b cells of the pancreas to the glucose signal . It also increased the sensitivity of the progressive long - term effect of asanas . Therefore, it was also found that brief yoga - based relaxation training normalizes the functions of the autonomic nervous system15 . As shown in previous studies, the findings of the present study suggest that yogic exercises can improve stress and stress biomarkers in nursing students . However, follow - up studies will be needed to evaluate the effect on biochemical parameters of stress such as cortisol and blood glucose in nursing students . To my knowledge, this is the first study to demonstrate the effect of yoga exercise on the blood glucose level of healthy nursing students . Use of only female subjects meant that we could not make comparisons between males and females . The participants were not on a strict and uniform diet plan, and this may have influenced the study results . Despite these limitations, these findings suggest that cyclic yoga exercise can improve not only the score of life stress, but also the postprandial blood glucose level in nursing students.
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Higher animals possess distinct muscle classes that are specialised for certain tasks: the vertebrate heart pumps blood life - long without rest, smooth muscles ensheathing the gut propel food without voluntary control, and body muscles move in a precise, consciously controlled manner to enable body movements, body posturing and facial expressions . To optimally fulfill these different tasks, each muscle class requires distinct contractile, metabolic and electrophysiological properties . The molecular basis for these functional distinctions is generated during development and results in a dramatically different morphology for each of the three muscle classes . Smooth muscles are mononucleated and can be activated by a variety of neuronal, hormonal, autocrine / paracrine signals or changes in load and length . They are activated through electrical coupling after neuronal firing and show regular striations along their myofibrils . Each muscle fiber contains many, often hundreds, of nuclei and has a defined neuromuscular junction that triggers contractions . Each fiber houses many highly ordered myofibrils that are laterally aligned to form stereotypical cross - striations . In this review, we discuss recent progress on mechanisms of differential transcription and alternative splicing that instruct functional differences between muscle types . We focus on mammalian skeletal muscle and drosophila body muscle as the best understood model systems . Mammalian skeletal muscle fibers are historically classified as slow (type 1, red muscle) or fast (type 2, white muscle) fibers . They generally can produce higher forces than slow fibers, are glycolytic and fatigue rather quickly . In contrast, slow fibers produce lower forces, primarily use oxidative metabolism and are more fatigue - resistant (reviewed in). Each individual human skeletal muscle consists of many, often several hundred, muscle fibers with a characteristic fiber - type composition . For example, the extensor digitorum longus (edl) muscle in the foot is mainly composed of fast fibers, whereas the soleus muscle in the lower leg contains mainly slow fibers . However, the individual fiber composition of each muscle will adapt to exercise regime, such that the soleus muscle of a sprint athlete will incorporate more fast fibers as compared to that of a marathon runner, which will be slower . The different functional properties of skeletal muscle fiber types in mice arise during fetal muscle development and are further modified during postnatal life . The general myogenic transcription factors myod, myf5, mrf4 and myogenin are required for the correct development of most, if not all, skeletal muscles early in embryogenesis (reviewed in). Subdivision into distinct muscle fiber types arises during late fetal development in mice through initiation of the fetal myogenic program . It was recently shown that the expression of nuclear factor one x (nfix) switches the embryonic to the fetal program by repressing embryonic and activating fetal myogenic genes such as muscle creatine kinase (mck) or -enolase . This enables the next steps of fiber - type specification by the differential expression of additional transcription factors . The best studied factors are six1 and six4, which promote the fast fiber fate, together with their cofactor eya1 (). Their action is supported by the transcriptional repressor sox6, which represses slow genes in fast fibers . Together, this complex interplay between general and specific transcription factors establishes the typical fiber - type distribution at the end of murine fetal muscle development . Postnatally, muscle fiber - type distribution is significantly reorganized, coinciding with substantial muscle growth after birth . Neuronal innervation, together with calcium - calcineurin signaling, is a key player at this stage . Increased calcineurin signaling promotes the slow fiber fate, potentially through the downstream cooperation of mef2d with the transcriptional coactivator pgc-1, which induces the expression of slow fiber genes, such as myoglobin, or genes required for mitochondrial oxidative metabolism . Varying levels of neuronal activity, and thus calcineurin signaling, also promote the differential recruitment of nfat family members to the promoters of activity - dependent genes . An nfatc2/3/4 complex specifies transcription of fast fiber genes, while the nuclear import of nfatc1 driven by slow nerve activity redirects the complex to activate transcription of slow genes . As in embryogenesis, general muscle transcription factors cooperate with fiber type - specific transcription factors to achieve differential expression of fiber type - specific genes during adult muscle differentiation . The best - studied examples of differentially expressed sarcomeric components in mammalian body muscle are the myosin heavy chain (myhc) isoforms . Different fiber types express different myhc isoforms from the various muscle myosin ii genes in mammals . During the embryonic to fetal myogenic switch is lost and type 2a fast fibers express myhc-2a, while slow fibers express myhcbeta/ slow (reviewed in). Myhc expression is at least partially regulated by nfat family members downstream of neuronal activity, as myhc - slow is cooperatively controlled by all four nfat family members, while myhc-2a is controlled by nfatc2/3/4 . While further details of upstream regulation are unclear, the expression of myhc isoforms with different molecular properties, for example variable cross - bridge lengths with actin during contraction, underlies part of the functional differences between fiber types (reviewed in). Additionally, specific myhc isoforms are combined with fiber type - specific isoforms of the troponin tropomyosin complex to adjust the calcium sensitivity of different fiber types . As a consequence, slow fibers already start contracting at low cytoplasmic calcium concentrations; whereas, fast fibers require higher calcium levels to initiate contraction and show a steeper tension increase upon further calcium influx due to a larger cooperativity in their calcium response . However, the physiological differences between fast and slow fibers depend on more than just differences in myosin, troponin or tropomyosin isoforms . A recent proteomics analysis in mice compared the slow soleus muscle with the fast edl muscle and identified 551 proteins that vary significantly between the two muscles . Most notably, these differentially expressed proteins were enriched for proteins involved in contraction, ion homeostasis, glycolysis and oxidation, emphasizing their roles in the different physiologies of these muscles . Despite this significant progress, our mechanistic understanding of how a combination of transcription factors and signaling molecules assembles functionally distinct muscle types and regulates such a broad repertoire of cellular genes remains limited . This is in part due to the high complexity and plasticity of the mammalian system: every muscle consists of many muscle fibers of different fiber types and is patterned by a complex interplay between autonomous and non - autonomous inputs on its transcriptional machinery at various stages during embryonic and adult development . Complementary to vertebrate studies, research in drosophila has added valuable insights into how different body muscle types are generated during development . Tubular muscle fibers in the head, legs, thorax and abdomen are responsible for most movements of the fly . Tubular muscles are very similar to vertebrate skeletal muscles, as they contain laterally aligned myofibrils that lead to a typical cross - striated pattern (fig . Nerve stimulation results in calcium release, which triggers actomyosin contraction and thus leads to a synchronous stimulation - contraction pattern . In stark contrast, drosophila indirect flight muscles (ifms) are asynchronous muscles that require stretch - activation in addition to calcium stimulation to induce their contractions . This stretch - activation mechanism allows very fast (200 hz) ifm oscillations, coupled with high force production (up to 80 w / kg muscle), enabling flight . Calcium does not cycle during contractions, but instead remains at an elevated level during the entire flight period due to continuous low frequency nerve stimulation . Ifms have high mitochondrial content for longlasting oxidative metabolism, little sarcoplasmic reticulum, and a fibrillar stretch - sensitive organization of their myofibrils (fig . How the striking morphological and physiological differences between tubular and fibrillar muscle arise during development can be mechanistically investigated with modern drosophila genetics . The drosophila tool kit includes systematic, tissue - specific loss of function studies with genome - wide rnai approaches using recently established transgenic genome - wide rnai libraries . The binary gal4-uas system allows tissue - specific gene knockdown at a given developmental time period, e.g. During pupal stages, thereby preventing pleiotropic phenotypes . A systematic muscle - specific rnai screen identified more than 2000 genes with a putative role in muscle, about 300 of which are required for normal flight behavior and thus likely function in ifms . Detailed morphological analysis of the ifms upon knock - down of these 300 genes identified the conserved zinc - finger transcription factor spalt major (salm) as a muscle - type specific selector gene for the fibrillar muscle fate . In salm knock - down animals, the fibrillar flight muscles are morphologically transformed to tubular, leg - like muscles (fig . Salm is expressed specifically in fibrillar muscle during development and, if misexpressed, is sufficient to switch tubular muscle to the fibrillar fate (fig . 1 g), making it a bone - fide muscle - type selector gene . Importantly, this function of spalt is not restricted to drosophila, but is conserved over at least 280 million years of evolution in most flying insects . Interestingly, spalt - like (sall) proteins are conserved to mammals and some sall family members are expressed in the mammalian heart . The heart is a very stiff muscle, whose contraction is also stretch - modulated, a phenomenon described as the frank starling mechanism, which links cardiac ejection to cardiac filling with stronger ejection upon larger filling . Mutations in sall1 lead to townes - brocks syndrome, a multi - organ syndrome that includes heart abnormalities, suggesting an evolutionarily conserved role of sall family members in stretch - activated muscle . How does salm execute this switch from tubular to fibrillar muscle development and to what extent does salm control the characteristic properties of stretch - activated ifms? Salm is the founding member of the conserved family of spalt zinc - finger transcription factors, which play important roles during the development of many organ systems, such as the eyes, wings and trachea in insects . In mammals, spalt - like (sall) genes are similarly required for the development of various organs including the heart and for specification of the mouse inner cell mass - derived lineages . Sall proteins are localized to the nucleus in mammals and drosophila and have been reported to act as both transcriptional repressors and activators, depending on the system studied . Despite their crucial roles in organ development, specific dna binding motifs for most of the spalt family members, in particular for drosophila salm, remain elusive . In ifms, salm either directly or indirectly activates the expression of fibrillar muscle specific genes such as flightin (fln) or troponin c isoform 4 (tpnc4) and at the same time represses tubular muscle - specific genes like troponin c41 (tpnc41) or muscle protein 20 (mp20). Importantly, it has been shown that all of these proteins directly regulate the contractile properties of the ifms: tpnc4 is critical for stretch - activation of the myofibrils, whereas fln is required for proper assembly of the thick filaments in ifms contributing to their high stiffness, an essential mechanical property for stretch - activation . In addition, salm up - regulates a wide range of mitochondrial genes specifically in ifms, which mainly rely on oxidative metabolism, thereby adapting them to the high energy demand of flight . Thus, salm not only switches myofiber fate by changing the transcriptional profile of core sarcomeric genes, it also determines most of the physiological differences between tubular and fibrillar muscle fibers . It is poorly understood how salm achieves ifm - specific target gene expression, especially when considering that salm function is also essential to activate distinct sets of targets in eye, wing or trachea cells . One possible mechanism is that salm cooperates with or modifies the activity of the essential muscle - specific transcription factor mef2 at particular mef2-dependent enhancers in ifms . Since vertebrate mef2 family members are well - known to cooperate with bhlh factors of the myod family, as well as with hdacs and hats, to achieve different regulatory outputs in distinct muscle types, it is plausible that salm similarly cooperates with or modifies the activity of drosophila mef2 at particular mef2-dependent enhancers in ifms . Such cooperative interactions of mef2 to instruct spatio - temporally restricted expression of target genes have also been demonstrated with the fusion competent myoblast - specific transcription factor lame duck (lmd), the cardiogenic transcription factor tinman and the general mesodermal factor twist . Mef2 is also required for ifm differentiation, being essential for proper formation and maturation of contractile filaments . However, whether cooperative interaction of salm with mef2 at enhancers of fibrillar muscle genes occurs awaits further investigation . Salm may additionally act downstream or cooperatively with the homeodomain proteins homothorax (hth) and extradenticle (exd). Despite broad expression in adult tubular leg and abdominal muscle as well as fibrillar ifms, adult muscle - specific loss of hth or exd results in a specific transformation of ifms to tubular muscles, and mis - expression of hth or exd in jump muscle, another large tubular muscle in the thorax, notably, while ifms express high levels of salm, wild - type jump muscle expresses low levels of salm; thus, it is attractive to hypothesize that the fibrillar fate results from transcriptional cooperativity of salm with hth and exd, which in wild type only occurs during development of the fibrillar ifms . These examples suggest that the cooperation of general myogenic transcription factors with muscle - type specific transcriptional regulators constitutes a basic principle for muscle fiber - type specification conserved from drosophila to vertebrates . Interestingly, the fibrillar ifm fate is not only determined by a transcriptional switch, but also by a change in the splicing pattern between fibrillar and tubular muscle . Mf is a muscle - specific thick - filament associated protein conserved in insects that is putatively involved in the assembly of thick filaments . Muscle - specific rnai - mediated knock - down of mf is pupal lethal, supporting an important role in fiber assembly . In drosophila, the mf locus generates multiple gene isoforms, including a short isoform that is specifically expressed in ifms and longer isoforms that are expressed in tubular muscles . Mrna sequencing of ifms, salm knock - down ifms as well as tubular leg and jump muscles reveals that the major difference between long and short mf isoforms is a regulated splicing event joining exon 5 with 6 in fibrillar muscles or exon 5 with 7 in tubular muscles (fig . The exon 56 junction is preferentially used in fibrillar ifms, whereas this splice event rarely occurs in tubular muscles or salm knock - down ifms (fig . Although two specific mf splicing events in larval muscles are regulated by muscleblind (mbl), the splicing factor regulating the switch from exon 56 to 57 splicing in fibrillar versus tubular muscle remains to be identified . In addition to mf, several other structural genes are reported to be alternatively spliced between tubular and fibrillar muscle, including drosophila troponin - t (upheld, up), tropomyosin 1 (tm1), myosin alkali light chain (mlc1), myosin heavy chain (mhc) (for review see) and projectin (bent, bt), a drosophila titin homolog . In salm knock - down animals, splicing of all these genes is likewise switched to the tubular instead of the fibrillar pattern, consistent with the hypothesis of a functional transition in the alternative splicing landscape to that normally found in tubular instead of fibrillar muscle . In general, inclusion or exclusion of particular coding sequences due to alternative splicing likely leads to the production of proteins with different regulatory or biophysical properties . While the functional significance of long versus short mf isoforms remains to be determined, aberrant splicing of drosophila troponin t (tnt, up) in ifms results in functional impairment (flightlessness) caused by defects in the myofibrillar apparatus, with diffuse z - lines and the formation of so - called these morphological and functional defects are presumably due to changes in tnt activity by differential phosphorylation of c - terminally located alternative exons, which affects tnt function and sarcomere stability . Another target of extensive splicing regulation is the large drosophila titin homolog, projectin (bt). Projectin is one of the largest drosophila proteins composed of 47 annotated exons, including multiple exons encoding a conserved proline - glutamic acid - valine - lysine (pevk)-rich domain . Interestingly, the pevk domain from vertebrate titin was shown to be elastic in vitro and in vivo, likely due to entropic changes upon stretch, suggesting a spring - like function during sarcomere contraction cycles . This implies that a difference in pevk domain length between fiber types may modulate the stiffness of vertebrate muscle . Indeed, vertebrate heart contains shorter titin isoforms with shorter pevk - rich domains resulting in an increased passive stiffness of the adult heart compared to skeletal muscle (; reviewed in). Mice with a deletion of the short pevk domain of the heart - specific isoform display a hypertrophic cardiomyopathy most likely caused by the changed elastic properties of titin . The drosophila titin - like protein projectin also displays elasticity and is proposed to function as a molecular spring . Although titin's fiber - type specific splice events might not be directly conserved between insects and vertebrates, ifms do possess a high passive resting stiffness that is required to facilitate stretch - activation with minimal sarcomeric displacement . Ifms normally contain the shortest observed pevk domain of only a few amino acids, and presumably a shift to one of the longer 363 or 464 amino acid domains found in tubular muscle would decrease passive resting stiffness and likely disrupt the stretch - activation mechanism powering flight . A final example of alternative splicing directly affecting sarcomeric function is drosophila myosin heavy chain (mhc), the motor protein that produces contractile force in muscle, which in contrast to vertebrates is encoded by the single mhc gene in drosophila . Alternative splicing generates the wide diversity of mhc protein isoforms expressed in drosophila muscle fibers . Ifms express a different complement of alternatively spliced exons than larval mhc isoforms, resulting in mhc proteins with distinct physiological properties . In particular, the relay domains encoded by exon 9 variants result in variations in mgatpase activity and actin sliding velocity and affect myofibril assembly and stability, while variants in the converter domain encoded by exon 11 affect caatpase, mgatpase, and actin sliding velocity . Thus, alterations in mhc splicing in ifms would affect myofibril assembly and stability and also change the fine - tuning of the myosin atpase and actin filament motility . Taken together, these three examples strongly suggest that muscle fiber - type selector genes switch not only the transcriptional but also the splicing status of the muscle to ultimately control muscle fiber - type specific morphological and physiological properties . Regulation of splicing in muscle is not limited to insects and is also well documented in vertebrate muscle . Mef2 family members modulate the differentiation of many tissues including muscle, and mef2d produces a unique muscle - specific splice isoform, mef2d2 . While the broadly expressed mef2d1 isoform inhibits expression of late muscle differentiation genes due to phosphorylation by pka followed by association with corepressors, the muscle - specific isoform mef2d2 escapes pka phosphorylation due to exon switching and thus can recruit the transactivator ash2l to activate transcription of late muscle targets . Vertebrates have three troponin t (tnt) genes encoding cardiac, slow and fast tnt isoforms . The fast tnt gene undergoes extensive muscle fiber - type specific splicing to produce different isoforms that affect its ca sensitivity and thus regulate actomyosin interactions . These examples strongly suggest that isoform switching is a basic principle in both vertebrate and drosophila muscle to fine - tune muscle fiber - type specific functional properties . Proper control of the splicing machinery is relevant for muscle function in mice and humans, as mis - regulation of splicing can lead to major muscle and heart diseases . While strong evidence supports the involvement of splicing factors such as muscleblind - like and rbfox in muscular dystrophies, the relationship between rna - binding motif protein 20 (rbm20) and titin in heart disease is perhaps the most provocative link to - date between alternative splicing and muscle disease . As discussed above, titin is the elastic component of the myofibril, maintaining the precise structural arrangement of thick and thin filaments and generating passive muscle stiffness . Alternative splicing of titin's pevk region modifies the spring property, with shorter, stiffer versions typically found in the adult vertebrate heart . Mutations affecting titin splicing that reduce passive stiffness are associated with dilated cardiomyopathy (dcm) and account for nearly one third of all cases of familial dcm . Titin splicing is at least partially regulated by rna - binding motif protein 20 (rbm20), which promotes exon skipping by repressing particular splicing events, normally resulting in the shorter titin isoforms expressed in the heart . Mutation of the rbm20 gene results in mis - splicing, notably resulting in the expression of longer titin isoforms (fig . 2d), which results in symptoms of dilated cardiomyopathy including ventricular enlargement, arrhythmia, extensive fibrosis and increased rate of sudden death in both rat and human . Moreover, rbm20 regulates conserved splicing events in at least 30 additional genes, including the enigma protein zasp / cypher, that are involved in sarcomere organization and ion transport in the sarcoplasmic reticulum, several of which are also associated with dcm . This example illustrates that correct alternative splicing is critical to muscle - type physiology and function . Muscle fiber - type specification is a complex process controlled by coordinated regulation of both transcription and alternative splicing . Research in model organisms such as flies provides an important complement to vertebrate studies in understanding the basic principles contributing to the development of different muscle - types . In the case of the evolutionarily conserved fibrillar selector gene salm from drosophila, mechanisms of transcriptional regulation of not only sarcomeric components but also genes dictating the physiologic status of the muscle, in addition to pervasive regulation of alternative splicing, are likely relevant to the underpinnings of cardiomyopathy in humans . While recent studies have started to provide insight into how functional and morphological differences are generated during development, many important questions await detailed clarification . One important challenge is to generate a complete network of both transcriptional and splicing regulators expressed in different muscle types, as factor cooperativity and feed - forward or feed - back mechanisms are important in fine - tuning muscle fiber - type specific physiological properties . The differences in physiological properties between fiber - types are functionally dictated by the biophysical properties of alternatively spliced forms of signaling and structural proteins . Thus, characterizing first which alternate protein isoforms are present in each fiber - type and second how inclusion or exclusion of particular protein domains affects muscle physiology will be essential to understand muscle fiber - type specific function . The ultimate goal is to molecularly define how different fiber fates are specified during normal development and how these fiber - types instruct the construction of physiologically different molecular machines . These insights may be applied for treating disease symptoms such as muscle wasting and cachexia, or to increase muscle fatigue resistance for occupational reasons.
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Pelvic pain results from many causes such as primary dysmenorrhea, uterine anomalies, menstrual outflow obstruction, endometriosis, myoma and adenomyosis . A 15-year - old nulligravida young woman with a history of severe intermittent pelvic pain presented a 4 - 5 centimeter mass . Laparotomy revealed left rudimentary horn, non - communication was confirmed by postoperative hysterosalpingogram (hsg) and magnetic resonance imaging (mri). Rudimentary horn should be considered in differentiation of pelvic pain and mass in young females . Dysmenorrhea is categorized as primary when no pelvic pathology is revealed and secondary when there is a known pathological source . Characteristics of secondary dysmenorrhea include beginning of pain 7 - 14 days before menstruation, continuation of pain after menstruation, resistance to non - steroidal anti - inflammatory drugs and contraceptive pills . In case of secondary dysmenorrhea, uterine and vaginal anomalies, menstrual outflow obstruction, endometriosis, adenomyosis and uterine myoma are considered in the differential diagnosis (1). Some of the congenital abnormalities in the mullerian duct present with dysmenorrhea including imperforated hymen, cervical atresia, transverse septum of the vagina and rudimentary horn besides unicorn unite uterus . In imperforated hymen, complete absence of the cervix might result in pain and obstruction for which hysterectomy is necessary (3). The incidence of mullerian duct malformation in the general population is estimated to be 43% while that of unicornuate uterus is about 0.4% (4). In another study ultrasonography performed two months ago, revealed a mass of 4.5 centimeters in her left adnexa . Another sonography described the mass as a 5 cm solid tumor in favor of uterine myoma on the left side . Expert sonography of this center and color doppler velocimetry reported a 5.3 centimeters myoma on the left side with high vascular flow suggesting leiomyosarcoma or degenerated myoma . Surgical findings revealed bilateral normal appearing ovaries . On the left side there was a solid mass with the fallopian tube connected superior to this mass and in a lower position in comparison to the right side . A rudimentary horn was considered . Thus, a small incision was made on the mass extracting about 2 cm of chocolate fluid compatible with old blood . Exploration of the cavity by a narrow metal applicator, showed a small cavity without an outflow pathway . At this stage, blue dye transit from fallopian tubes occurred without entry to the peritoneal cavity . Due to no bilateral entrance of dye, while passing from tubes, technical problems was not ruled - out . In the operative field non - communication of the rudimentary horn biopsy of rudimentary mass and cavity was done for pathological review and surgical intervention was not taken . The treatment plan was the use of contraception while hysterosalpingography and mri were performed to achieve definite diagnosis before the next surgery . Unfortunately the patient rejected surgery due to post - surgical pain relief resulting from evacuation of chocolate fluid . Pathological report of mass biopsy suggested a " vascular myoma " and cavity biopsy reported " inactive endometrium " . Hysterosalpingography (hsg) report indicated a right unicornuate uterus with normal right fallopian tube and normal peritoneal spillage . The other side of the rudimentary horn and fallopian tube was not shown by the hsg examination . The mri report about 1.5 month after the operation revealed a 4 cm mass on the left side of the uterus with a small central cystic blood - containing locus of about 1.1 cm . The patient accepted a third operation after menarche and resection of rudimentary horn and left fallopian tube was done about 10 months after the second surgery, due to recurrence of severe pain . Uterine anomalies are rare and they are seldom considered in differential diagnosis of patients with clinical presentation of pelvic pain (6). In the present case, uterine myoma was considered as the first diagnosis and uterine anomalies were not mentioned in differential diagnosis . Most rudimentary horns are asymptomatic due to their non - functional and non - communicating pattern (3). In the case of endometric development in rudimentary horn, menstrual fluid is produced and implantation of conception product might occur . Thus, rudimentary horn pregnancy and pelvic pain occur . Rudimentary horn pregnancy mimics the clinical presentation of ectopic pregnancy resulting in surgical emergency (7). Ultrasound revealed an empty uterus and free fluid in the abdominal cavity . In the laparotomy procedure, rudimentary another case report of ruptured rudimentary horn pregnancy at 19 weeks was by hassan et al . Rudimentary horn pregnancy is visually miss - diagnosed until the occurrence of rupture and emergency . There is a case report on an asymptomatic 33-year - old pregnant woman who was diagnosed by laparoscopy - hysteroscopy following the diagnosis of ectopic pregnancy . In the laparoscopy, this case cooperated due to hysteroscopic and pre - rupture diagnosis . In our case, intraoperating dye injection was done, as well, although definite diagnosis was not done . As mentioned earlier, diagnosis of rudimentary horn is not made until the reproductive age when ruptured rudimentary horn pregnancy or pelvic pain occur . Pain is due to endometrial response to hormonal activities after menarche . In the present case, her history included several surgical interventions and miss diagnosis with appendicitis and other entities . In the rare case of unicornete non - communicating horn, surgical resection is necessary to relief pain and prevent rudimentary horn pregnancy and resulting emergency situations (10). Surgery was done more than two years after menarche as the third surgical intervention . In the first surgery diagnosis was appendicitis due to pain . After the second surgical intervention definite diagnosis was made . Due to transient pain relief after the second surgery, the patient and her family rejected definite surgery until she experienced severe pain again within 10 months . Intraoperative limitation of case management (in the second surgery) was inability to show open tube on the right side, thus definite diagnosis and surgery at that stage was not possible . Hysteroscopy was acceptable, although it was not done due to the inability to diagnose this case . Rudimentary horn should be considered in cyclic pelvic pain starting after menarche besides a mass like lesion, sometimes mistaken by myoma . Early diagnosis is needed to relief pain and prevent surgical emergency by rudimentary horn resection.
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Pome has unfavourable environmental ramifications effects including land and aquatic ecosystem contamination and loss of biodiversity and increase in cod and bod in environment [1, 2]. Today, the penetration of palm oil mill has been considered due to its effluents into the waterways and ecosystems remaining a meticulous concern towards the food chain interference and water consumption . This can cause considerable environmental problems if discharged without effective treatment by polluting land and effectively suffocating other aquatic life . Thus, palm oil mills are required to treat their pome prior to discharging it into rivers and streams . In the process of palm oil milling, pome is mainly generated from sterilization and clarification of palm oil in which a large amount of steam and hot water are used . Pome is a thick brownish liquid that is compiled with high concentrations of total solids, oil and grease, chemical oxygen demand (cod), and biological oxygen demand (bod). The biological treatment depends enormously on consortium of microorganism's activities, which operate the organic substances present in the pome as supplements and eventually degrade these organic matters into simple by - product such as methane, carbon dioxide and hydrogen supplied, and water . The biological treatment process requires large pond to hold the pome in place for the effective biodegradation, which regularly takes a few days relying upon the sort and native of the microorganisms . Besides, so as to enhance the effectiveness of this medication process, powerful mono or combined culture of feasible fungi and bacteria in biodegradation treatment of pome waste . Therefore, the challenge of converting pome into an environmentally friendly waste demanded an efficient treatment and effective disposal technique . A variety of microorganisms have been investigated to be capable of biodegrading oil wastewater with high profits . Anaerobic and aerobic treatments are the great and capable biological methods for pome treatment . However, the suspended and colloidal components are neither effectively decomposed biologically nor by other conventional means because their floating on the surface of the wastewater has an impact on the microbial cycle . Particularly, the major component in biological digestion, microorganism plays an important role and core factor of the system to control reactor performance and stability . This research intended to investigate the viability of microorganisms present in pome under conditions for treatment . The process is investigated in terms of cod removal and decolorization (admi) with ph tolerance . This experimental work carried out on the activated sludge processes would give some accepting treatment of the microbial behaviour as well as the substrate removal performances and evaluation of the application of an existing palm oil waste on digestibility of pome condition . Experiment was conducted in 250 ml erlenmeyer flasks that contained 100 ml of pome that was covered with cotton gauze stopper . To study the effectiveness of microorganism's type on pome treatment, the medium was inoculated with inoculums culture (10% v / v) containing approximately 10 cfu / ml of mixture of the isolated microorganisms (bacteria and fungi) in separate flasks and incubated at 37c on an incubator shaker (150 rpm) for 7 days . The flasks containing the pome samples were autoclaved at 121c and 15 psi for 15 min to produce the autoclaved samples . Both the autoclaved and raw flasks were inoculated with 5% (v / v) of the prepared inoculums . Three parameters were investigated in this study, whereas the effects of bod, cod, ph, and admi were investigated (figures 1, 2, 3, and 4). Raw pome sample was collected by grabbing a sample directly from the inlet of a waste stabilization pond which discharged from stone and sand filtration from local industry in johor bahru province in malaysia . Sample was collected in clean containers, was directly transferred to the department of biotechnology and medical engineering laboratory, university technology malaysia (utm), and was stored at 4.0c in the laboratory cold room for further treatments . The effluent was prefiltered by means of simple depth filtration to remove the coarse solid found in the suspension and experimental procedure for effect of microbe growth on the pome reduction . Pome samples were collected from open pond system which is the most popular treatment method and is utilized by more than 85% of mills . About 0.1 ml of pome sample was serially diluted in sterile distilled / deionized water and aliquots of the dilutions were ascetically plated into the media (nutrient agar and nutrient broth, resp . ). The agar plates were incubated at 37c for 2448 hours to enumerate the aerobe and facultative bacteria and the fungi culture plates were incubated and inverted at 37c for 37 days . After incubation, the colonies that grew on the medium were counted and expressed as colony morphological and physiological characteristics . The dilution / isolation streaking procedure involves the dilution of bacteria by streaking them over the agar surface to obtain isolated cells which will subsequently grow into isolated colonies . A total of 70 facultative aerobe colonies were isolated from pome and cultured on two different media, nutrient agar, and nutrient broth . It is found that potential of treatment of pome was that ten of the colonies had the ability to reduce the cod and decolorization . Characterization and identification of aerobes to genus level using biochemical tests based on tables 3 and 4 . The characterization of pome microorganisms that are subcultured were identified by comparing their characteristics with biochemical tests . Identification of fungi was based on microscopic morphology and cultural characteristics and the bacteria isolates that were characterized based on biochemical test . The derived characteristics were compared with those known using manual of determinative bacteriology and isolated bacteria were compared with the culture characteristics . Fungi identification was based on the macroscopic and microscopic morphology . For the microscopic morphology, a drop of ethanol was placed on a clean slide with the aid of a sterile needle, a fragment of the pure culture was transferred into the ethanol on the slide, and a drop of lacto - phenol blue stain was added and the ethanol was allowed to evaporate . The bacteria isolated from the pome included aspergillus niger, aspergillus flavus, mucor sp ., fusarium sp ., penicillium sp ., staphylococcus aureus, and bacillus sp . Inoculated into the autoclaved sample and then inoculated sample based on isolated microorganisms was incubated (10 cfu / ml) at 37c and agitated at 150 rpm . The pellet from the centrifugation of pome was used to determine the growth of the bacterial consortium . Two ml of distilled water was added into the pellet and was mixed homogenously using pipette . They were then transferred into the cuvette and the growth was measured using spectrophotometer at 600 nm wavelength . To separate the fungal biomass and the liquid medium, the whole fungi culture was centrifuged at 4,000 rpm for 15 min at 4c . Analytical parameters of pome were analyses based on the methods developed and modified by the department of environment of malaysia, doe, such as cod (digestion method), bod (biochemical oxygen demand), and the color (admi unit), which were determined according to the apha method . The analysis of cod was carried out according to the oxygen equivalent of the organic material in wastewater . Preliminary determination of cod usually relies on the amount of organic pollutants found in water and wastewater . The remained supernatants from each of the samples taken from three - hour intervals were used to determine the cod . One ml of the supernatant of each treated sample was transferred into cod vial and it was diluted with 0.9 ml distilled water (cod 01500 ppm range) and the mixture was mixed gently . The vial was placed in the preheated cod reactor at 150c for 2 hours . The percentage of cod removal was determined by cooling it at room temperature for 1 hour and determined by the spectrophotometry method . The absorbance was read at 620 nm (cod 01500 ppm range using hach program). For the blank, the color was determined by the american dye manufacturers institute (admi) method; the light scan absorbance was from 400 to 700 nm using shimadzu uv-160a, uv - visible recording spectrophotometer . Thus, 0.1 ml of the supernatant was diluted with 3.9 ml of phosphate buffer . They were mixed homogenously prior to transfer into the cuvette to be quantified of their degree of decolorization . The quantification was done by measuring the reduction of optical density (od) at 471 nm . Biochemical oxygen demand (bod) test is a widely used parameter for evaluating the ability of naturally occurring microorganisms to digest organic matter, in 37 days incubation at 37c by analyzing the depletion of oxygen inside the pome . Bod is the most commonly used parameter for determining the oxygen demand on the receiving water of a municipal or industrial discharge . Bod can also be used to evaluate the efficiency of treatment processes and is an indirect measure of biodegradable organic compounds in water . A measured amount of the biodegradation sample was poured into the 300 ml bod standard bottle . The result from this study shows that the microbial species isolated are similar to those found in local pome . However, pseudomonas species, staphylococcus aureus, and bacillus species, which are bacillus sp ., pseudomonas sp ., and staphylococcus sp ., spores make pome microbial species to be inactive and highly resistant to lethal effect of boiling, dry heating, and ultra violet radiation . Lipase accelerates in hydrolysis of lipid causing consequent breakdown into fatty acid and alcohol [23, 24]. Aspergillus niger and aspergillus flavus have been distinguished for their ability to endure oily wastewater such as pome . Shows that these fungi are able to survive in hostile environment [21, 22, 25]. The value of ph recorded in through this study is lower, that is, more acidic than the ohimain et al . . The low ph of the pome indicates that it is acidic and should be treated to reach 77.5 ph as indication level of plant compatible . The acidic character of pome may have been influenced by the corrosion of iron used in processing . When the pome is discharged into the soil or stream, it affects nutrient availability of the nearby plants; in addition, most plants grow within a ph range of 6.57.5 . Decolorization is one of the vital parameters in pome quality assessment and indicates the physical and biological processes prevailing in the pome; it points to the degree of pollution in wastewater [29, 30]. The major predominate microorganisms in pome have been variously mobilized for the treatment of the wastewater [3134]. During degradation, oil and grease in the pome are broken down and mineralized . The oily environment may provide a good environment for lipolytic microorganisms to flourish due to the unrecovered oil present in the effluent . However, the microbial content of pome is a good indicator of biodegradability of wastewater and it could also be used in bioremediation of hydrocarbon from crude oil spills . However, since most of these organisms are spore formers, it helps them to survive the harsh environmental conditions of pome such as acidity [37, 38]. Moreover, the dark brown color of palm oil mill effluent is composed of organic compounds such as anthocyanin and carotene pigment that were extracted from fresh fruit bunches in the sterilization process . Furthermore, it included polyphenol compounds, tannin, polyalcohol, and melanoidin [39, 40]. The cod removal for the biodegradation of pome samples is depicted in figure 1 . In general there was gradual decrease in the cod concentrations of the raw pome sample including the inoculated multimicroorganism which was selected in this study and the control . The control sample tendency was observed for the biodegradation which was used as the noninoculum under controlled which is suspected to be undergoing some biochemical synthesis in 7 days . Furthermore, it was observed that the removal of cod in the autoclave sample was very close to the raw sample and this may suggest inoculated sample with isolated microorganism in the raw sample . In addition, the activities of local microorganisms may be used to describe the degradation of raw pome with high degradation of pome . The ph of the entire biodegradation samples increased from acidity towards alkalinity; processes increase alkalinity though at different rate and dimensions (figure 2). The control raw (raw cntl) sample and those inoculated with selected microorganism, respectively, reached neutral point at the end of the period and one important point to note is that treatment should reach to high standard of ph of pome for releasing through the environment . At the other end is a point of maximum expected in cod and bod reduction with proper fungi and bacteria in biodegradation of pome . A ph of 7.6 could vastly increase the benefits, since the treatment of the pome is for plant reutilization and discharge into river streams with less effect on the environment . Furthermore, the ph of the autoclaved samples is the case in the study which was normally below the ph of the unautoclaved samples except for sample with microorganism treatment . Moreover, close relationship was observed in the trend of ph changes between the raw and autoclaved pome sample inoculated with privilege of selected microorganism; however, treatment could accelerate the biodegradation in process . The changes in the biochemical oxygen demand (bod) of the samples were checked out for seven days (164 hours) in the bod incubator (figure 3). The concentrations of the bod generally decreased with numbers of days for all the samples inoculated with multimicroorganisms as well as the control sample but treatment shows stability in final stage . This trend is positive to the biodegradation treatment process of high strength wastewater such as pome, because the decrease in the bod indicates the reduction in the environmental organic load on the receiving water stream . The changes in the color of the samples were checked out for seven days (164 hours) based on the admi method at 471 nm (figure 4). The concentrations of the color generally decreased with numbers of days for all the samples inoculated with multimicroorganisms as well as the control sample but treatment shows stability in final stage, whereas there are fluctuation for control group which indicates contradiction in metabolites of microorganism system (growth between 6071 hours and 132 hours). This treatment shows positive tendency in biodegradation treatment of high strength wastewater such as pome, because the decrease in the color indicates the reduction in the environmental organic as a control application . It is really an indication that colorization is a vital parameter for determining biodegradation which directly signifies the metabolic status of probably the most delicate microbe group within the biodegradation system . The cod and bod reduction performance was monitored each 12 hours in batch system consisting of multibacteria isolated from pome wastewater . Table 3 shows the result for cod and admi values after treatment by using single bacteria in batch system each 12 hours in 7 days . The adaptation of the mesophilic microorganism operating condition at 37c led to a stable process that is significantly different from that previously found at 37c (table 2). This could be attributed to the rapid development of biodegradation wastewater that was originally present in the mesophilic sludge to become dominant under the new degradation conditions based on isolated local microorganisms . The persistent fungi were aspergillus niger, aspergillus flavus, aspergillus fumigates, fusarium, penicillium, mucor, and candida and bacteria were pseudomonas, micrococcus, staphylococcus aureus, and bacillus . This could be due to the fact that a substantial amount of these organisms may be eliminated from palm fruits when processing into the oil or contaminated after discharge into pond . High population of fungi in the pome may be associated with contaminations from poor sanitation in the mills . Moreover, it may also be due to the treatment process and the existing environmental conditions in the mills . The microbial species found in pome has the potential of degrading hydrocarbon in the wastewater . Biodegradation is associated with the saprophytic ability of fungi to grow on and degrade carbon sources in industrial effluents and reduce factor which could influence the environment . In this study, the acidic tendency of pome treatment (table 2) may affect nutrient availability of the nearby plants which pasture from discharge of pome in river; final ph reached approximately 7.6 (ut raw) in appropriate range rather than raw - cnt and auto - cntl (6.5 and 5.4, resp .) (figure 2); most plants grow and do better within a ph range of 6.57.5 . Bod is an important parameter in pome quality measurement and indicates the physical and biological processes surpass in the pome biodegradation; it indicates the degree of pollution in wastewater, and bod value obtained during the study was in the range of 155000168000 mg / l (figure 3). The effect of different oxygen concentration on the reduction of cod was depicted in figure 1 . It implies that the population of microorganisms plays a vital role in the process of reduction of cod, thus purged wastewater . Figure 1 showed that the number of bacteria increases and at the same time causes the reduction in cod concentration of pome, approximately 12000 mg / l in 168 hours of treatment . Figure 4 presents the colorization which shows positive tendency in biodegradation treatment of high strength pome; color measurement indicates the reduction in the environmental organic 486 to 180 admi . The result showed that all the samples collected from various mills have similar microbial species . The bacteria isolated from the pome include aspergillus niger, aspergillus flavus, mucor sp ., fusarium sp ., penicillium sp ., staphylococcus aureus, and bacillus species inoculate into the autoclaved sample (10 cfu / ml) incubated in flask in stable incubator . The growth of the potential isolated aspergillus fumigatus and aspergillus niger were determined at different temperatures . Substantial growth was observed at temperatures 3537c . In the case of growth at different condition with mixture of local microorganism, environmental issues have been gradually increased to becoming one of the most important in economic activities . In addition, the physicochemical conditions of pome from palm oil mills was studied . The following parameters: ph, admi, bod, and cod, the synergistic effect of bacteria and fungi, for pome treatment, brings about enhanced performance for effective biodegradation . Bod, cod, and od values obtained during the study were in the range of 155000168000 mg / l, 12000 mg / l, and 180 admi, respectively (figures 1, 3, and 4) and ph was tending toward neutral point (figure 2) contrary to previous studies (table 2). The viability of using isolated microorganisms for effective biodegradation of pome has been investigated in this study and it can be deduced that the digestion of pome samples based on local isolated fungi and bacteria . This result indicates the potential of isolating of indigenous microorganism that will be effective in the degradation pome; on the other hand, the isolated microorganism requires nutrient expansion for the effective biodegradation of pome . Due to the high concentrations of bod and cod in the oil palm processing effluents, discharging it into the environment could cause pollution; hence, it is preferable to recycle rather than discharging it . By improving environmental condition of living microorganisms in a biodegradation process, substrate - related factors could be utilized in bringing up a flourishing achievement in aerobic waste degradation process . The addition of functional inoculum and selected high effective indigenous microorganisms is expected to save the pome degradation time and cost for reduction of bod, cod, and color.
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Although adenovirus (adv) infection usually causes self - limiting respiratory disorders in immune competent children; severe and systemic adv infection in children undergoing chemotherapy for leukemia has been continuously reported . Nevertheless, there has been no consensus on risk factors and treatment strategies for severe adv infection in children undergoing chemotherapy . We report a case of a 15-year - old boy with a fatal systemic adv infection . He had received reinduction chemotherapy for relapsed acute lymphoblastic leukemia under continuing antifungal therapy for previously diagnosed fungal pneumonia . He complained of fever and right shoulder pain 4 days after completing the reinduction chemotherapy . In spite of appropriate antibiotic and antifungal therapy, a multiplex polymerase chain reaction test for respiratory viruses was positive for adv in a blood sample, and a urine culture was positive for adv . He received oral ribavirin, intravenous immunoglobulin, and intravenous cidofovir therapy; however, he eventually died . Relapsed leukemia, concurrent fungal pneumonia, and delayed cidofovir administration were considered the cause of the grave outcome in this patient . Adv may cause severe infections not only in allogeneic hematopoietic cell transplant recipients, but also in patients undergoing chemotherapy for acute leukemia . The risk factors for severe adv infection in patients undergoing chemotherapy should be determined in the future studies, and early antiviral therapy should be administered to immune compromised patients with systemic adv infection . Adenovirus (adv) infection usually causes self - limiting respiratory disorders in immune competent children . However, the severity of adv infection varies according to adv serotype, and higher severity and poorer prognosis have been observed in immune compromised children compared with immune competent children . Among children receiving allogeneic hematopoietic cell transplantation (hct), adv viremia occurred in 6% to 28% of cases, and 13% to 50% of patients with adv infection died . Meanwhile, the incidence of adv infection is relatively low in children receiving anticancer chemotherapy for acute leukemia . Adv reportedly causes less than 5% of respiratory viral infections in these patients, and few die from adv infections . We saw a case of fatal systemic adv infection in a child with acute lymphoblastic leukemia (all). The patient received reinduction chemotherapy for relapsed all under antifungal therapy for previously diagnosed fungal pneumonia, and was infected by adv during neutropenia following the reinduction chemotherapy . Although the patient did not receive allogeneic hct, relapsed all and concurrent fungal pneumonia in the patient might have led to the grave outcome of his adv infection . A 15-year - old boy complained of fever and right shoulder pain after reinduction chemotherapy for relapsed all . He was diagnosed with all at another hospital 3 years and 9 months prior, and was transferred to our hospital 2 months prior . At that time, he received reinduction chemotherapy for his second relapse of all; however, there was no response, and he received another course of reinduction chemotherapy during this hospitalization . The patient had been diagnosed with possible invasive pulmonary aspergillosis (ipa) during a previous hospitalization and had received oral voriconazole (4 mg / kg twice a day) therapy for 3 weeks following liposomal amphotericin b (5 mg / kg / day) therapy for 2 weeks . On the fever day (fd) 1, blood tests revealed a white blood cell count of 50/mm (neutrophils 0%, lymphocytes 0%), hemoglobin concentration of 8.3 g / dl, platelet count of 53000/mm, erythrocyte sedimentation rate of 34 mm / h, c - reactive protein level of 8.70 mg / dl, and serum galactomannan level of 0.21 . Chest x - ray images showed a round consolidation on the right upper lung fields, similar to the finding observed on admission (fig . The dose of voriconazole was increased to 7 mg / kg twice daily because his serum voriconazole level was <0.5 g / ml . Empirical antibiotics were changed to meropenem and teicoplanin due to persisting fever on fd 3 . Although his serum voriconazole level was 8.6 g / ml and serum galactomannan level was 0.14 on fd 6, the fever persisted . Chest computed tomography (ct) imaging performed on fd 6 showed an aggravation of the pneumonic consolidation on the right upper lobe compared with the chest ct images used for the diagnosis of fungal pneumonia during the previous hospitalization (fig . The antifungal agent was changed to liposomal amphotericin b (5 mg / kg / day) because we considered that the previously diagnosed possible ipa might actually be mucormycosis . However, his fever persisted, and gross hematuria developed on fd 12 . We suspected adv infection based on the concurrent hematuria and aggravation of the respiratory disease identified on the chest x - ray images (fig . A multiplex polymerase chain reaction (pcr) test for respiratory viruses was positive for adv in a blood sample . The pcr test was qualitative with no report on viral dna titer, but the cycle threshold was 26.64 for positive pcr results . Urinary bk virus, cytomegalovirus, and other bacteria were not identified; however, a urine culture was later reported to be positive for adv . On fd 15, oral ribavirin (10 mg / kg 3 times daily) rather than cidofovir was initiated, as the patient had not received allogeneic hct and his general condition was relatively stable except for his prolonged fever . However, the fever and gross hematuria did not resolve, and chest x - ray images showed an aggravation of the pneumonia without respiratory symptoms . Chest ct performed on fd 21 showed further aggravation of the pneumonic consolidation on the right upper lobe and a new consolidation on the right lower lobe accompanying pleural effusion (fig . 2c). We considered superimposing adv pneumonia and proposed cidofovir therapy; however, his parents did not accept this treatment due to its high costs . Intravenous immunoglobulin (ivig) of 1 g / kg was administered for 3 days instead of cidofovir; however, repeated multiplex pcr of blood was positive for adv, with a cycle threshold of 14.92 on fd 28, and his fever persisted . Although additional ivigs (150 mg / kg / day) were administered for 5 days, the fever persisted, his serum creatinine level increased to 2.36 mg / dl and oliguria developed . On fd 33, all antibiotic and antifungal agents were stopped due to acute kidney injury, and bilateral percutaneous nephrostomy was performed . His serum creatinine level began to decrease; however, the fever did not resolve and chest x - ray images showed further aggravation of the pneumonia (fig . Although the first dose of cidofovir (5 mg / kg) was administered on fd 36, the patient complained of dyspnea and tachypnea, and required oxygen therapy on fd 38 . On fd 40, multiplex pcr of a blood sample was again positive for adv, with a cycle threshold of 13.41, and liposomal amphotericin b, which was stopped due to acute kidney injury, was readministered . Mechanical ventilation was applied on fd 45, and he died on fd 48 . A bronchial wash fluid culture from a sample collected on fd 46 was reported as positive for rhizopus spp . And adv after his death . Chest x - ray images showing round consolidations on the right upper lung fields upon admission (a), insignificant change in the consolidation on fever day 1 (b), and aggravation of the pneumonic consolidation on fever days 12 (c) and 35 (d), respectively . Chest computed tomography showing pneumonic consolidation with surrounding ground glass opacity on the right upper lobe during the previous hospitalization (a), and aggravation of the pneumonic consolidation on fever days 6 (b) and 21 (c), respectively . Weekly monitoring with quantitative pcr for adv in blood is recommended in allogeneic hct recipients at risk for adv infection, and preemptive antiviral therapy with cidofovir is recommended for patients positive for adv viremia . However, routine monitoring for adv infection in autologous hct recipients and leukemia patients receiving chemotherapy is not necessary due to its lower incidence and severity compared with allogeneic hct recipients . Respiratory viral infections occur in 43% to 59% of children undergoing chemotherapy or those with febrile neutropenia; however, severe complications due to respiratory viral infections are rare and mortality due to respiratory viral infections is only 0% to 2% . Adv comprises only 2% to 5% of respiratory viral infections in immune compromised children, less frequent than rhinovirus and respiratory syncytial virus infections: thus, the clinical impact of adv infection has been underestimated . However, severe and systemic adv infection in children undergoing chemotherapy for leukemia has been continuously reported . Children with severe adv infections tend to have low absolute lymphocyte counts; however, severe adv infection may occur during any period of the scheduled chemotherapy, including induction, consolidation, and maintenance chemotherapy . Nevertheless, there has been no report on risk factors for severe adv infection in children undergoing chemotherapy . Future studies are required to elucidate the risk factors for severe adv infection in children undergoing chemotherapy, and appropriate strategies for early diagnosis and antiviral treatment for children most at risk should be established . Although no specific antiviral therapy has been established for adv infection, cidofovir is recommended as the first - line agent for adv infection in allogeneic hct recipients . Ribavirin is a broad - spectrum antiviral agent; however, its antiviral mechanisms have not been exactly defined . Because a significant efficacy of ribavirin was not observed in allogeneic hct recipients with systemic adv infections, it is not recommended as a therapeutic agent for adv infections in allogeneic hct recipients . However, its clinical effectiveness in allogeneic hct recipient with hemorrhagic cystitis due to adv has been reported . Systemic adv infections, which occurred in a patient with myelodysplastic syndrome, who had not received hct, and in solid organ transplant recipients, responded to ribavirin therapy . Our patient, who had not received allogeneic hct, was relatively clinically stable upon diagnosis of adv viremia, although he complained of fever and gross hematuria . At that time, we administered ribavirin rather than cidofovir because there are currently no guidelines for treatment of adv infection in children undergoing chemotherapy rather than hct . In addition, cidofovir may cause adverse effects, such as kidney injury and bone marrow suppression, and increase hospital cost . Although cases showing the ivig effect on systemic adv infections in solid organ transplant recipients were reported, its effect on adv infections of immune compromised patients has not been evaluated in controlled studies . Moreover therefore, ivig is not recommended as a therapeutic agent for adv infections as well as ribavirin . Although our patient's clinical status and viremia worsened after ribavirin therapy, cidofovir therapy was delayed for 3 weeks after the diagnosis of adv viremia due to parental hesitation in deciding to administer cidofovir . Despite administration of cidofovir, 43% to 50% of allogeneic hct recipients with adv viremia die: early administration of cidofovir reportedly improves the prognosis of adv infections . Although our patient had not received allogeneic hct and t - cell - specific immune suppressants, uncontrolled underlying all, concurrent fungal pneumonia, and delayed cidofovir administration could have led to the grave outcome . Fatal adv infections in allogeneic hct recipients with concurrent bacterial and fungal coinfections have been reported, although the adv dna titers from blood samples were low . In conclusion, adv may cause severe infections not only in allogeneic hct recipients, but also in patients undergoing chemotherapy for acute leukemia . Adv infection should be considered in immune compromised patients with severe respiratory tract infections or hemorrhagic cystitis . The risk factors for severe adv infection in patients undergoing chemotherapy should be determined, and early antiviral therapy should be administered to immune compromised patients with systemic adv infection.
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The immune system has evolved to protect the host from infections and cancer . Typically, the immune system is divided into two categories- innate immunity and adaptive immunity . The innate immune system comes into play immediately after the appearance of antigen whereas the adaptive immune system provides antigen - specific response . In addition to these defense mechanisms, there are unconventional t cells like the gamma delta () t lymphocytes and natural killer t (nkt) cells that functionally and phenotypically belong to both the innate and the adaptive immune system and are able to bridge the two123 . In the peripheral circulation of humans, t cells comprise about 1 - 10 per cent of the circulating t cells, though this percentage can rise to as high as 50 per cent at some mucosal sites4 . T cells are involved in combating infectious diseases and have non - redundant capacities in the inhibition of tumour development and progression56 . Unlike t cells, t cells do not require the help of conventional major histocompatibility complex (mhc) class i and class ii molecules for recognizing the antigens1 . Antigen recognition by t cells is dependent upon the particular variable (v) region of the t cell receptor (tcr) as opposed to the entire rearranged tcr required by t cells . T cells expressing v1 are abundantly found at mucosal sites and these respond to the expression of non - classical mhc molecules on the surface of virally - infected or tumour cells789 . V2 + (v9v2) cells are predominantly present in the peripheral circulation and respond to non - peptide phosphoantigens1011 . V9v2 t cells recognizes self and microbial phosphorylated metabolites generated in the eukaryotic mevalonate pathway and in the microbial 2-c - methyl - d - erythritol 4-phosphate (mep) or non - mevalonate pathway12 . It was observed that during bacterial and protozoan infections, v9v2 t cells expand to high levels which in some individuals represented the majority of circulating t cells13 . Subsequent characterization of the microbial antigens recognized by human t cells revealed that these are non - proteinaceous in nature and have critical phosphate residues1116 . The v9v2 crystal structure confirmed the presence of a basic, positively charged region in the binding groove that could directly interact with the negatively charged pyrophosphate moiety of the antigen10 . These phosphoantigens are generated during the non - mevalonate and mevalonate pathways utilized by prokaryotic and eukaryotic cells, respectively121718 . Various compounds like steroid hormones, cholesterol, many types of vitamins, rubber, etc . Are derived from this pathway . There are now many synthetic phosphorylated compounds that are capable of stimulating t cells like bromohydrin pyrophosphate (brhpp), 4-hydroxy-3-methyl - but-2-enyl pyrophosphate (hmbpp) and mono - ethyl pyrophosphate141920 . In addition to phosphoantigens, there are reports on additional ligands for human t cells . Bisphosphonates, especially nitrogen - containing bisphosphonates (nbp) are widely used to treat postmenopausal osteoporosis and skeletal malignancies . Inhibit the key enzyme farnesyl pyrophosphate synthase (fpps) of the mevalonate pathway, thereby upregulating the pool of endogenous ipp . The accumulated ipp activates v9v2 t cells to release inflammatory cytokines interferon (ifn)- and tumour necrosis factor (tnf)-. Another class of molecules that stimulate v9v2 t cells is alkylamines . These are also present in edible plant products such as tea, wine, apples and mushrooms21 . T cells discriminate transformed tumour cells from healthy cells by the upregulation of self - antigens like heat shock proteins (hsp). The expression of these proteins are increased in tumour cells due to higher metabolism and serves as endogenous danger signals623 . Studies from our laboratory have demonstrated that v9v2 t cells recognize hsp60 on oral tumour cells and have the ability to lyse autologous and allogenic oesophageal tumour targets via recognition of hsp60 and hsp702526 . The influx of tils (tumour infiltrating lymphocytes) to the tumour site enhances the potential for anti - tumour immune responses . The numbers and types of lymphocytes present in the infiltrate are related to the chemokines produced by both the tumour cells and tissue stromal cells located at the tumour site . For example, breast, cervix and pancreatic tumours as well as ovarian tumour produce cc and cxc chemokines that are important mediators of macrophage and lymphocyte infiltration in those tumours272829 . Interestingly, both v1 and v92 t cells display distinct chemokine receptors that bestow these cells the property to migrate to the tumour site . V1 in addition, v92 t cells express nk receptor p1a (nkr - p1a) platelet endothelial cell - adhesion molecules (pecam) while v1 use nk receptor p1a nkr - p1a for transendothelial migration31 . V1 t cell subsets from the peripheral blood utilize a larger array of adhesion molecules, namely lfa-1, vla-4, vla-5, l - selectin and e7, to bind to squamous cell carcinoma cells compared to the restricted usage of lfa-1, l - selectin and cd44v6 by the v2 t cells32 . The mutually exclusive pattern of chemokine receptor expression in both the subsets of t cells indicates independent mechanism of homing to tumour site that might have an important aspect in cancer immunotherapy . Ability of t lymphocytes to produce abundant proinflammatory cytokines like ifn-, potent cytotoxic effector function and mhc - independent recognition of antigens makes it an important player of cancer immunotherapy . T cells kill many different types of tumour cell lines and tumours in vitro, including leukemia, neuroblastoma and various carcinomas33343536 . Accumulation of mevalonate metabolites in tumour cells is a powerful danger signal that activates the t cells . In normal cells, ipp produced by mevalonate pathway are at a concentration that is insufficient to trigger t cells response . However, dysregulation of mevalonate pathway in certain tumours leads to production of higher concentrations of ipp, which is sensed by tcr as a tumour antigen3738 . It was also shown that mrna knockdown of ipp - consuming enzyme, fpps, induced v9v2 t cell stimulation in otherwise non - stimulatory tumour cells39 . T cells are able to recognize and kill many different differentiated tumours cells, either spontaneously or after treatment with different bisphosphonates, including zoledronate . It has been shown that human tumour cells can efficiently present aminobisphosphonate and pyrophosphomonoester compounds to t cells, inducing its proliferation and ifn- production40 . Combination treatment utilizing v9v2 t cells along with chemotherapeutic agents and zoledronate has been shown to induce an increase in the cytotoxic function of t cells against solid tumour4142 . The ability of t cells to efficiently kill bisphosphonates treated colon cancer stem cells and ovarian cancer stem - like cells has also been reported3643 . In addition to phosphoantigens, t lymphocytes can also be activated by mitochondrial f1-atpase - related structure expressed together with apolipoprotein a - i, which are expressed on the surface of some tumour cells44 . F1-atpase displays characteristic of antigen presentation molecule by binding to the adenylated derivative of ipp and promoting tcr aggregation, cytokine secretion and cytotoxic activity45 . Natural killer (nk) receptors expressed on t cells play a crucial role in mediating the anti - tumour response of t cells . Natural killer group 2, member d protein (nkg2d) expressed on v9v2 t cells is critical for tumour recognition and provides activation signals upon binding to non - classical mhc molecules of the mhc class i chain - related molecules (mic) and ul-16 binding protein (ulbp) families expressed on tumour cells464748 . This ligand binding to nkg2d can affect the release of tnf-, interleukin (il)-2 receptor (cd25) upregulation and increase cytolytic potential of t cells47 . Ulbp molecules are involved in v9v2 t cells recognition of leukemias and lymphomas49 and also ovarian and colon carcinomas50 . T cells utilizing the v1 chain isolated from tumour - infiltrating lymphocytes can also kill cancer cells . V1 t lymphocytes have been shown to mediate cytolytic activity by recognizing mica, micb or ulbp expressed on cancer cells5152 . T cells resemble nk cells as these also express cd16 (fcriii) receptor . Upon recognition of phosphoantigens, a subset of v9v2 t cells upregulates cd1653 . It has been reported that cd16 represent activation / memory status of t cells and these cd16 cells have specific phenotypic features that distinguish these from the cd16 subset . These constitutively express several natural killer receptors (nkg2a / cd94) and high amounts of perforin, but express low levels of chemokine receptors (cxcr3, ccr6) and ifn-54 . Cd16/fcriii receptor binds to fc portion of immunoglobulin g (igg) and engagement of cd16 by t cells leads to antibody - dependent cellular cytotoxicity (adcc)55 . Adcc is a process in which cd16 + effector cells actively lyse tumour cells that have been bound by specific antibodies . Several reports have proven that in vitro t cells respond to activation via cd16 and mediate adcc against tumour with therapeutic anti - tumour monoclonal antibodies (mabs) like rituximab, trastuzumab, of atumumab and alemtuzumab355657 . It has also been shown that stimulated t cells increase the efficacy of trastuzumab in vivo in her2 + breast cancer patients58 . Given the potent antitumour effector function of t cells and broad reactivity to many different types of tumours has raised a great interest to explore their therapeutic potential . An important feature of t cells is that these favourably kill cancer cells and show low (if any) reactivity towards non - transformed cells which makes these very good candidates for cancer immunotherapy50 . The safety and efficacy of t cell - based immunotherapy have been evaluated in several clinical trials59 . Presently, two strategies for t cells in tumour immunotherapy have been applied . These are the adoptive cell transfer of in vitro expanded t cells and the in vivo therapeutic application of -stimulating phosphoantigens or aminobisphosphonates together with low - dose recombinant il2 (ril2). Studies carried out in nude mice demonstrated that repeated infusion of t cells leads to tumour growth arrest60 . Another study carried out in scid mice showed the anti - tumour effector functions of nk cells and t lymphocytes against autologous melanoma cells61 . In one pilot study, patients with b - cell malignancies that failed conventional therapy were treated with intravenous administration of pamidronate and ril2 to stimulate v9v2 t cells in vivo62 . It was observed that in vivo v9v2 t cells were expanded in five out of nine patients; three out of these five responding patients had partial remissions and one had stable disease . Other trials with adoptive transfer of t cells include patients with advanced cancer like metastatic renal cell carcinoma63 and non - small cell lung carcinoma64 where stable disease was found in 60 and 37 per cent patients, respectively . In these cases, the regimen consisted of ex vivo activation and expansion of autologous v9v2 t cells with either phosphoantigens, such as brhpp or aminobsphosphnates, like zoledronate or pamidronate or their infusion into the patients . Aminobisphosphonates have also been used in clinical trials to treat metastatic prostate cancer65 and advanced breast cancer66 where partial remissions have been reported . Complete remission of lung metastasis in a patient with renal cell carcinoma has also been reported after adoptive transfer of t cells67 . It was shown that the patient was disease free for two years without any additional treatment following in vitro activation and expansion of autologous t cells with hmbpp plus ril2, combined with the infusion of zoledronate and ril267 . There is also increasing evidence that stimulating effector t cells can enhance monoclonal antibody - induced cytotoxicity and thereby improve the anticancer effects of mabs . It was found that repeated infusions of phosphoantigens stimulated t cells and trastuzumab increased the efficacy of t cells against her-2 breast carcinoma cell lines in vivo58 . In addition, a survival advantage to patients with an increased t cells following allogeneic stem cell transplantation (asct) has been reported . A long - term survival advantage in a group of high - risk acute leukemia patients who recovered with increased number of circulating t cells following partially mismatched related haematopoietic stem cell transplantation was reported68 . The unique features of human t cells related to antigen recognition, tissue tropism, lack of antigen processing requirement and cytotoxic function make these ideal candidates for cancer immunotherapy . T cells recognize increased pool of endogenous ipp (a consequence of dysregulated mevalonate pathway) in cancer cells, release ifn-/tnf- and mediate cytolyic effector functions . Expression of nkg2d receptors provides a selective advantage to t cells to recognize tumours that express stress induced molecules like mica / b . This property of t cells can be exploited for immunotherapy as tumours downregulate mhc molecules to evade immune recognition (fig . ). One way to exploit t cells for cancer immunotherapy is the use of synthetic phosphoantigens like brhpp or hmbpp which can act as tcr agonists . Future trials should harness bisphosphonate activated t cells in combination with chemotherapy or monoclonal antibodies for treatment of solid tumours and haematologic malignancies . Mechanism underlying t cell killing of tumours: t cell receptor (tcr) interacts with isopentenyl pyrophosphate (ipp) generated through the mevalonate pathway in tumours . Bisphosphonates inhibits farnesyl pyrophosphate synthase (fpps) leading to increased endogenous pool of ipp and dimethylalleyl pyrophosphate (dmapp) in tumour cells . T cells recognize heat shock proteins (hsps) and mhc class i chain - related molecules (mica / b) or ul-16 binding protein ulbp expressed on tumour cells via their tcr and natural killer group 2, member d protein (nkg2d) receptors, respectively . T cells can also kill tumour cells through antibody dependent cellular cytotoxicity (adcc). T cells expressing cd16 (fcriii) interacts with tumour associated antigens (taa) via specific monoclonal antibodies and mediate adcc . Cytokines like interferon- (ifn-) and tumor necrosis factor- (tnf-) released by t cells can recruit other immune cells (bystander effect).
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Lack of functional ability in the upper extremities after stroke restricts usage, and causes asymmetric posture and contracture in daily life, thus exacerbating functional limitations of the upper limbs1 . The functional recovery period of the upper limbs in patients with hemiplegia is estimated to be 11 weeks after stroke . However, if there is no functional improvement with intensive treatment in this period, it is difficult to expect better prognosis or improvement2 . Previous studies demonstrate that applying kinesiology tape in the same direction as the muscle fibers facilitates muscle function3, while perpendicular application inhibits muscle function4 . However, there is no report on the influence of taping with respect to a neurophysiologic background . Thus, additional in - depth quantitative studies about the effects of taping on balance, gait, injury prevention, strength, range of motion, and proprioception are required to confirm its clinical efficacy . Therefore, this study determined the effects of kinesiology taping on upper - extremity function and activities of daily living (adl) in patients with hemiplegia . This study involved an alternative medicine intervention in order to demonstrate whether taping improves movement dysfunction after stroke . Thus, the results will provide fundamental data for the application of this technique in clinical practice . This study was conducted from june 4 to december 22, 2012 on 30 post - stroke patients who were hospitalized in a rehabilitation hospital in chungcheong province, south korea . The patients in the experimental group (eg, n = 15) and control group (cg, n = 15) performed task practice with and without 3ns kinesiology taping, respectively . The inclusion criteria were as follows: post - stroke hemiplegia, at least 6 months since onset, and ability to communicate and understand instructions . Meanwhile, patients with peripheral neuropathy, musculoskeletal disease, or dementia were excluded . All patients provided written informed consent to participate in the study prior to its commencement . The intervention was performed for 30 minutes, 3 times per week for 28 weeks for a total of 84 sessions . The patients practiced tasks as per the researcher s instructions, while sitting on a comfortable chair . The task practices included wiping a table with a towel, putting a block into a box, and stacking cups . Taping of the deltoid muscle taping of the quadratus lumborum muscle for the anterior aspect of the middle deltoid, the patient extended their arm and performed slight external rotation; then, the therapist placed the tape on the region from the humerus prominence to the coracoid process . For the posterior aspect of the middle deltoid, the patient placed the affected side of the hand on the opposite side of the acromion process, and the tape was placed on the region from the humerus prominence to the lateral third of the scapular spine . For the lateral portion of the quadratus lumborum, the tape was applied from the posterior superior iliac spine to the 12th rib while the trunk was bent laterally to the unaffected side . For the medial portion of the quadratus lumborum, the tape was placed from the posterior superior iliac spine along the transverse processes of the lumbar vertebrae while the trunk was bent forward . Upper - extremity function and adl were assessed by using the manual function test (mft)5, modified motor assessment scale (mmas)6, brunnstrom recovery stage7, and functional independence measure (fim)8 . Chicago, ll, usa). The test was used to analyze the general characteristics of the patients . Because the kolmogorov - smirnov test indicated the data were not normallydistributed, the wilcoxon signed - ranked test and mann - whitney u - test were used to determine intra- and intergroup differences, respectively . The level was set at = 0.05, and the level of significance was set at p <0.05 . The eg comprised 8 women and 7; 8 and 7 patients had right and left hemiplegia, respectively . Meanwhile, the cg comprised 9 women and 6 men; similar to the eg, 8 and 7 patients had right and left hemiplegia, respectively . The mean ages of the eg and cg were 69.20 7.07 and 67.33 9.50 years respectively . There were no significant differences in the general characteristics between groups . However, the mft and mmas results differed significantly after the intervention in both groups (eg: 22.47 6.55 and, 13.87 5.06; cg: 21.33 6.23 and, 13.80 5.25, respectively). However, there were no significant differences between groups pre- or post - intervention . There was no significant change in the brunnstrom recovery stage of the hand after the intervention in either group, but the meanof post - intervention score tended to be higher in the eg . However, post - intervention, the fim changed significantly within each group and differed significantly between groups (table 1table 1.changes in upper - limb function and activities of daily living (n = 30)mftmmasbrshfimprepostprepostprepostpreposteg (n = 15)16.40 5.8522.47 6.55 9.87 4.3713.87 5.065.13 1.305.27 1.3383.73 16.03 90.53 16.65cg (n = 15) 19.80 6.0521.33 6.23 12.60 5.19 13.80 5.255.40 1.245.40 1.24 77.13 16.6678.40 16.62p <0.01 within a group, p <0.05 between groups . Eg: experimental group; cg: control group, mft: manual functional test; mmas: manual motor assessment scale; brsh: brunnstrom recovery stage of the hand; fim: functional impairment movement). Eg: experimental group; cg: control group, mft: manual functional test; mmas: manual motor assessment scale; brsh: brunnstrom recovery stage of the hand; fim: functional impairment movement a previous study evaluating the effects of kinesiology taping indicates that applying tape to the affected upper limbs of post - stroke patients reduces spasms, resulting in improved range of motion, strength, and function9 . Another study reports that muscle recruitment patterns can be altered because of better proprioception by stimulating mechanoreceptors10 . In the present study, this indicates that task practice positively influenced upper - extremity function, regardless of taping . This suggests that applying tape can improve the restricted coordinated movement of joints in the upper extremities because of dysfunction of normal movement reflex, diminished coordination, and integration of motor control and disconnection between the muscles in the upper extremities and trunk; this results in improved upper - limb movement . Because adl involve interactions among several elements such as joints, muscles, sensory inputs, and nerves, the assessment of adl is crucial to determine the condition of patients with a brain injury11 . In the present study, this indicates that applying tape to the shoulder and lumbar region improves postural control and shoulder girdle stability . However, there was no significant difference in the brunnstrom recovery stage of the hand either within each group or between the groups . This demonstrates that taping does not affect the control of muscle tone or coordination in the distal part of the upper extremities . Furthermore, limited ability in manipulation did not improve prior to the recovery of the movement and stability in the proximal part of the upper limbs . Even if patients with hemiplegia can reach with their arm to grasp an object, they cannot do so because of an inability to manipulate the object using their hands . Thus, they have more restrictions for using the affected upper limb and will therefore have fewer opportunities to use it12 . This may be why patients with hemiplegia use support for their disabled hand or are unable to use it at all . Consequently, such patients experience many life difficulties after their stroke, because the functions of the upper limbs and hands are vital for adl . Future studies should perform surface electromyography to verify that muscles affected by taping regain strength after the intervention.
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Urea cycle disorders (ucd) are among the most common inborn errors of metabolism of the liver . Citrullinemia can be classified into three types: neonatal / infantile (types i and iii) and the adult form (type ii). More than 20 patients with citrullinemia have undergone liver transplantation, with favorable therapeutic results . However, the number of cases with auxiliary partial orthotopic liver transplantation (apolt) is limited . Apolt was initially performed in patients with acute liver failure and non - cirrhotic metabolic liver disease to compensate for enzyme deficiencies without complete removal of the native liver and for small - for - size living donor grafts [3 - 5]. Our patient was a 27-year - old man who was diagnosed with adult - onset type ii citrullinemia (ctln2) at age 24 . The patient had been treated with a low protein diet and oral administration of lactulose . Recently, the patient has had more frequent alterations of his mental status with irritability and altered consciousness . Even though oral administration of l - arginine, kanamycin and intravenous hyperalimentation with branched chain amino acids was started, his plasma levels of ammonia and citrulline were highly elevated, up to 844 g / dl (normal range, 19 to 87 g / dl) and 682 mol / l (normal range, 12 to 55 mol / l), respectively . Despite the conservative medical treatment including alternative pathway medication, intermittent continuous renal replacement therapy and hemodialysis, his condition failed to improve . Therefore, living donor liver transplantation was planned using a right lobe graft from his 25-year - old sister . However, the estimated volume of the donor's right lobe was 800 ml compared to 180 ml of the left and in addition there was a type iii portal vein anomaly . We eventually decided to perform an apolt using the extended left lobe of the liver from the donor . The actual graft volume was 200 g and the graft - to - recipient weight ratio (grwr) was only 0.33% . The patient's extended left lobe (including caudate lobe) was resected and the extended left lobe graft (including middle and left hepatic vein) was transplanted orthotopically . The left and middle hepatic vein trunk of the extended left lobe graft was anastomosed to the left and middle hepatic vein trunk of the recipient in end - to - end fashion with interposition venoplasty using the recipient's greater saphenous vein as a patch . The left portal vein of the graft was anastomosed to that of the recipient in end - to - end fashion . Considering that the graft was too small, the recipient's right portal vein was narrowed by surgical clipping, instead of total diversion, for prevention of the development of portal venous hypertension . The left hepatic artery of the graft was anastomosed to that of the recipients in end - to - end fashion . Intraoperative doppler ultrasonography demonstrated that portal and arterial blood flow were well maintained in both the native liver and the graft . On postoperative day 7, abdominal computed tomography demonstrated orthotopically transplanted small left lobe graft with well - enhanced pattern (fig . The post - operative course was uneventful and the patient was discharged without any problems . The plasma concentrations of ammonia were at the upper limits of normal, so only a low protein diet (50 g / day) was supplied without any specific medication . On post - operative day 33, the patient reported a sudden severe visual disturbance and the plasma levels of ammonia were noted to have increased up to 209 g / dl . The estimated computed tomography volume of the graft was decreased to 190 ml and a left portal vein stenosis was suspected . Immediate coil embolization of 8th segmental branch of right anterior portal vein and self - expandable stent placement for left portal vein was performed in order to increase the portal flow to the graft (fig . Stent insertion day 7, the calculated computed tomography volume of the graft was increased to 250 ml (fig . Apolt was initially introduced as a temporary or permanent support for patients with fulminant hepatic failure . Apolt can provide an adequate hepatic mass to correct underlying enzyme deficiencies in adult patients with ucd . This procedure can be carried out to overcome the limitations of graft volume in living donor liver transplantation . In korea, therefore, living donor liver transplantation is the only treatment option for patients with metabolic liver disease such as ucd . In this case, the graft weight of 200 g and the grwr of 0.33% are the smallest graft and lowest margin ever reported to meet the minimum requirements for the demands of adequate metabolic function in adults . Because the remaining native liver function, except for the metabolism of ammonia, is normal, the differential diagnosis including portal steal phenomenon and rejection as well as other problems associated with the vascular system is difficult . Thus the plasma ammonia level is a valuable follow up marker and should be closely monitored in such patients . In addition, careful monitoring of the portal flow to the graft is important for the detection of delayed graft dysfunction . Selective portal vein embolization is a useful method for the increase of portal flow to the graft . In conclusion, apolt can be a life saving procedure and an effective treatment for patients with adult - onset type ii citrullinemia, especially when a deceased donor graft is not available or when candidate donors have insufficient graft volume . However, further confirmation is needed with additional patients and long - term follow - up . How much volume of graft can be transplanted successfully is questionable in cases of inborn error of metabolism, such as ucd.
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An example of such complex equipment is a liquid chromatography - tandem mass spectrometry (lc - ms / ms) system, which, in recent years has become a frequent addition to clinical laboratories for a variety of applications including testing for drugs of abuse, therapeutic drug monitoring and steroid analysis. [36] such devices offer high potential sample throughput, but, as they are not currently accompanied by vendor - supplied interfaces to enable them to be connected to a laboratory information system (lis), they also impose a potentially burdensome requirement for manual data transfer with the concomitant risk of transcription errors . To the authors knowledge, the only commercially available software to software interface that exists for the mass spectrometer in use here, is from data innovations llc (south burlington, vermont, usa). The advantage of using a commercially available product is that the company already has software developed for a number of different instruments and the laboratory does not require expertise in computer programing to implement such an interface . However, the disadvantages are the significant cost of such an interface and the decrease in control and ease of adaptability of such a product as far as the laboratory is concerned . A group in the department of pathology at the university of pittsburgh medical center developed a data management interface to help solve this problem successfully interfacing a waters 2795 lc - ms / ms to a sunquest lis . We describe here the implementation of the same data management interface using the same lis, but a different mass spectrometer that is in use in our laboratory and we extend their work by describing the pre - processor code that was required to convert the data output from the mass spectrometer into a format accepted by the lis . The targeted analytical mass spectrometer was the ab sciex 5500 qtrap (ab sciex, foster city, ca) coupled to a shimadzu prominence uflc (ultra - fast liquid chromatography, shimadzu scientific instruments, pleasanton, ca) lc system . The standalone computer running the lc - ms / ms was a dell optiplex 960 that uses the microsoft windows 7 operating system (microsoft corporation, redmond, wa). The analyst 1.5.1 application (ab sciex, foster city, ca) acquires data from the mass spectrometer and generates a results table upon the user's request . A text file (txt) of the results table can then be exported from analyst by the user and saved in the appropriate folder . The data management software was licensed from the university of pittsburgh (automated file transfer for mim batch style interfaces; id01516). The data management software and the other support modules are written in autohotkey (ahk), a free open - source scripting language for automating activities on a computer operating under the windows operating system . Both the compiled executable programs and the source text files are included with the data management software . The software program was installed solely on the computer running the lc - ms / ms system . The lis utilized was sunquest (sunquest information systems inc ., tuscon, az). Standard parameters such as analyzer and assay identification codes as well as quality control (qc) definitions for the lc - ms / ms assay were defined by adding them to the sunquest maintenance, using the straightforward and well - documented methods provided by the vendor . The targeted analytical mass spectrometer was the ab sciex 5500 qtrap (ab sciex, foster city, ca) coupled to a shimadzu prominence uflc (ultra - fast liquid chromatography, shimadzu scientific instruments, pleasanton, ca) lc system . The standalone computer running the lc - ms / ms was a dell optiplex 960 that uses the microsoft windows 7 operating system (microsoft corporation, redmond, wa). The analyst 1.5.1 application (ab sciex, foster city, ca) acquires data from the mass spectrometer and generates a results table upon the user's request . A text file (txt) of the results table can then be exported from analyst by the user and saved in the appropriate folder . The data management software was licensed from the university of pittsburgh (automated file transfer for mim batch style interfaces; id01516). The data management software and the other support modules are written in autohotkey (ahk), a free open - source scripting language for automating activities on a computer operating under the windows operating system . Both the compiled executable programs and the source text files are included with the data management software . The software program was installed solely on the computer running the lc - ms / ms system . The lis utilized was sunquest (sunquest information systems inc ., tuscon, az). Standard parameters such as analyzer and assay identification codes as well as quality control (qc) definitions for the lc - ms / ms assay were defined by adding them to the sunquest maintenance, using the straightforward and well - documented methods provided by the vendor . Four discrete data are required to successfully transmit an individual patient result from the lc - ms / ms output text file to sunquest: the unique patient container identification number (cid - a barcode affixed to the patient sample container that is scanned by a barcode reader, electronically entering the cid into the lc - ms / ms software); the sunquest test code for the assay being performed (in this case test code ptes for pediatric total testosterone); the assay result for each patient and the qc data (the total testosterone concentrations obtained from running 3 qc materials with each batch of patient samples with mean concentrations of 9, 168 and 535 ng / dl respectively in this case). The data management software consists of libraries of code that are called from a primary compiled program that handles the various aspects of export file management and transfer to the lis via file transfer protocol (ftp). The documentation included with the licensed data management software was comprehensive; however, minor discrepancies were discovered between the documented installation file locations and the actual locations specified in the software source code (included with the software). Accurate placement of the library and resource files in appropriate directories and subdirectories was required for the compiled program to find them . Although the author was not familiar with ahk at the time of installation, its similarities to other scripting languages permitted casual review to catch these discrepancies . The documentation supplied with the data management software clearly describes the process of integrating a pre - processor, which is required if the data output format from the lc - ms / ms software does not match the input format of the lis, but is not supplied with the data management software . In this case, pre - processing of the text file exported from the analyst software was required in order for sunquest to accept the data and perl was chosen by the authors for writing the pre - processor . Perl is a popular and well - established open - source scripting language used in many contexts, including biomedicine, is freely available for multiple platforms and can be installed on windows platform (as well as others) via activeperl installer . Perl stands for practical extraction and report language and is highly suitable for parsing text, making it an ideal language to use for a pre - processor in the context of the analyst - sunquest interface . A particularly helpful feature of perl utilized extensively in the pre - processor is its implementation of regular expressions, which facilitates searching for particular patterns in a given text string and optionally replacing them with another string . Although the data management software includes a log file, we found that additional logging was helpful during the debugging process, and thus we added additional logging to the pittsburgh software source files, which were then recompiled . With this in mind, the pre - processor includes a separate log with output to verify that the various steps in the program occur each time it is run . A flow chart documenting the steps in the process the source code for the pre - processor is shown in figure 2, and an explanation of each line of the code can be found in table 1 . The workflow process from the analyst software to the pre - processor, the data management software and finally to the transfer by file transfer protocol to the laboratory information system source code written in perl to pre - process the text file output from the liquid chromatography - tandem mass spectrometry system to a format suitable for transfer to sunquest via file transfer protocol key to the purpose of each line of the source code that was used to pre - process the data output file from the liquid chromatography - tandem mass spectrometry system during the validation of the ptes assay, the interface was tested by exporting the text file containing calibrator, qc and patient assay results into a specific folder; in this case the report output folder on the computer running the mass spectrometer . The correct order of events is as follows: the text file containing the results will disappear from the report output folder; when the pre - processing has been completed, a text file will then be found in the converted folder; this file disappears from the converted folder and will then be found in the sent folder; the original text file exported from the lc - ms / ms system will then be placed in the archived folder . In order to determine if the interface was performing as expected, when the pre - processed file was found in the sent folder, the sunquest lis was accessed (after a delay to allow sunquest to perform its periodic scan of the stipulated upload location for the data) to determine if the qc results and patient assay results had indeed been transferred via ftp, and that the calibrator results were essentially ignored by the pre - processor . As this process was working successfully, it was implemented once the ptes assay was placed into clinical use . To date, there have been no software problems or failures . The interface has eliminated the need for manual data entry and any associated manual transcription errors . Four discrete data are required to successfully transmit an individual patient result from the lc - ms / ms output text file to sunquest: the unique patient container identification number (cid - a barcode affixed to the patient sample container that is scanned by a barcode reader, electronically entering the cid into the lc - ms / ms software); the sunquest test code for the assay being performed (in this case test code ptes for pediatric total testosterone); the assay result for each patient and the qc data (the total testosterone concentrations obtained from running 3 qc materials with each batch of patient samples with mean concentrations of 9, 168 and 535 ng / dl respectively in this case). The data management software consists of libraries of code that are called from a primary compiled program that handles the various aspects of export file management and transfer to the lis via file transfer protocol (ftp). The documentation included with the licensed data management software was comprehensive; however, minor discrepancies were discovered between the documented installation file locations and the actual locations specified in the software source code (included with the software). Accurate placement of the library and resource files in appropriate directories and subdirectories was required for the compiled program to find them . Although the author was not familiar with ahk at the time of installation, its similarities to other scripting languages permitted casual review to catch these discrepancies . The documentation supplied with the data management software clearly describes the process of integrating a pre - processor, which is required if the data output format from the lc - ms / ms software does not match the input format of the lis, but is not supplied with the data management software . In this case, pre - processing of the text file exported from the analyst software was required in order for sunquest to accept the data and perl was chosen by the authors for writing the pre - processor . Perl is a popular and well - established open - source scripting language used in many contexts, including biomedicine, is freely available for multiple platforms and can be installed on windows platform (as well as others) via activeperl installer . Perl stands for practical extraction and report language and is highly suitable for parsing text, making it an ideal language to use for a pre - processor in the context of the analyst - sunquest interface . A particularly helpful feature of perl utilized extensively in the pre - processor is its implementation of regular expressions, which facilitates searching for particular patterns in a given text string and optionally replacing them with another string . Although the data management software includes a log file, we found that additional logging was helpful during the debugging process, and thus we added additional logging to the pittsburgh software source files, which were then recompiled . With this in mind, the pre - processor includes a separate log with output to verify that the various steps in the program occur each time it is run . A flow chart documenting the steps in the process the source code for the pre - processor is shown in figure 2, and an explanation of each line of the code can be found in table 1 . The workflow process from the analyst software to the pre - processor, the data management software and finally to the transfer by file transfer protocol to the laboratory information system source code written in perl to pre - process the text file output from the liquid chromatography - tandem mass spectrometry system to a format suitable for transfer to sunquest via file transfer protocol key to the purpose of each line of the source code that was used to pre - process the data output file from the liquid chromatography - tandem mass spectrometry system during the validation of the ptes assay, the interface was tested by exporting the text file containing calibrator, qc and patient assay results into a specific folder; in this case the report output folder on the computer running the mass spectrometer . The correct order of events is as follows: the text file containing the results will disappear from the report output folder; when the pre - processing has been completed, a text file will then be found in the converted folder; this file disappears from the converted folder and will then be found in the sent folder; the original text file exported from the lc - ms / ms system will then be placed in the archived folder . In order to determine if the interface was performing as expected, when the pre - processed file was found in the sent folder, the sunquest lis was accessed (after a delay to allow sunquest to perform its periodic scan of the stipulated upload location for the data) to determine if the qc results and patient assay results had indeed been transferred via ftp, and that the calibrator results were essentially ignored by the pre - processor . As this process was working successfully, it was implemented once the ptes assay was placed into clinical use . To date, there have been no software problems or failures . The interface has eliminated the need for manual data entry and any associated manual transcription errors . We successfully implemented a licensed data management interface between sunquest and an ab sciex mass spectrometer that may allow the laboratory more control and adaptability than a commercially available software to software interface . Currently, this interface has only been used to transmit results from one test to individual patients, but the authors are confident in the adaptability of this interface in order to be able to handle more complex results, such as pharmacokinetic analysis consisting of a number of samples from the same patient over a given time period, each with a different concentration . Although the licensed software from the university of pittsburgh was extremely useful in facilitating an interface between the lc - ms / ms system and the lis, local expertise in programming was necessary to perfect the data transfer process.
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The vertebrate immune system employs a wide variety of antigen - specific receptors - the immunoglobulins and t - cell receptors - to recognize and neutralize foreign invaders . The receptor diversity necessary to recognize an almost limitless universe of potential pathogens is created by a site - specific dna rearrangement process termed v(d)j recombination . This unique reaction assembles the receptor genes from separate v, d and j gene segments, a process ostensibly restricted to lymphocytes at a certain stage of their development . In this view, the prediction is that the germline dna of a given organism should contain unrearranged receptor genes; rearranged versions of the genes should exist only in lymphocytes . This prediction was satisfied by examination of germline and lymphocyte dna samples from a variety of familiar vertebrates, including mice, humans, birds, and common farm animals . The fly in the ointment, however, appeared with an analysis of receptor genes in the germline of evolutionarily distant organisms - the cartilaginous fishes . In these primitive vertebrates, many of the immunoglobulin genes are actually found pre - rearranged in the germline (reviewed in). This suggests that the v(d)j recombinase may actually be an evolutionary force, a notion that is strongly supported by recent studies in the nurse shark . The evolutionary consequences of these site - specific germline gene rearrangements may reach far beyond the immune system . The recombination machinery recognizes dna sequences called recombination signal sequences (rsss) adjacent to each gene segment . Each rss consists of conserved heptamer and nonamer motifs separated by 12- or 23-nucleotide' spacer' sequences . The recombinase is made up of two proteins, rag-1 and rag-2, which, in conjunction with the non - specific dna - bending proteins, hmg-1 or hmg-2, recognize the rss and catalyze site - specific dna cleavage (see for review). Cleavage requires both rag-1 and rag-2, and co - expression of these proteins is thought to be limited to developing lymphocytes (, and reviewed in). As illustrated in figure 1, the rag proteins introduce a double - strand dna break precisely between the v, d or j coding sequence and the rss, generating two types of dna ends: blunt signal ends (which terminate in the rss) and covalently sealed (hairpin) coding ends (which terminate in the v, d or j element). After cleavage, the two signal ends are joined, producing a signal joint . Prior to joining the coding ends, the hairpins must be opened; joining generates a coding joint that may have lost or gained nucleotides . The precise details of the end - processing and joining reactions remain obscure, and are not important for this story; it is clear, however, that multiple, non - lymphoid - specific dna repair proteins are involved . One detail that is important is that the opening of the hairpins frequently occurs' off center', leading to palindromic single - stranded tails . Joining of these ends can give rise to a characteristic signature in the completed junction: a palindromic, or p nucleotide, insertion . Another type of junctional insertion, n (non - templated) nucleotides, are added randomly by the enzyme terminal deoxynucleotidyl transferase . Thus, coding joints formed by v(d)j recombination have several distinguishing characteristics, including variable loss of nucleotides and the frequent presence of either n nucleotides, p nucleotides, or both . V(d)j recombination bears many striking parallels to the movement of certain transposable elements, both in its general form and in important mechanistic details of the reaction (reviewed in). In fact, recent biochemical experiments have shown that purified rag proteins can catalyze transposition in the test tube, integrating a dna fragment bearing signal ends into a target duplex (figure 2). This reaction does not require specific dna sequences in the target and, like many transposition reactions, creates a characteristic' footprint': upon integration, three to five nucleotides of target dna are duplicated on either side of the transposon . It should be noted that there is, as yet, no firm evidence that rag - mediated transposition events can occur in living cells . Early on, it was suggested that the v(d)j recombination system might have arisen by the fortuitous integration of a transposable element into an ancestral antigen - receptor gene . This hypothesis was strengthened by the discovery that the rag genes are tightly linked, and by the finding that the rag proteins can act as a transposase . Thus, a plausible model for the acquisition of the v(d)j recombination system during vertebrate evolution is the integration of a transposable element carrying the linked rag genes into a primordial antigen - receptor gene in an ancestral jawed vertebrate, approximately 450 million years ago (reviewed in). Presumably, this initial integration event created the first rearranging antigen - receptor gene; subsequent gene duplication events then created the multiple immunoglobulin and t - cell receptor loci . To learn more about the evolutionary origins of the combinatorial immune system, several laboratories have characterized antigen - receptor loci from a wide variety of species, including the cartilaginous fishes - the living jawed vertebrates most phylogenetically distant from mammals . The immunoglobulin loci of sharks and skates contain some fairly typical antibody genes, with multiple v, d and j elements . Surprisingly, however, many of the immunoglobulin genes are already partially or fully rearranged in the germline (see for review). Recently, pre - rearranged immunoglobulin genes were also found in a teleost fish, the channel catfish . One possibility is that they are descendants of the ancestral antigen - receptor gene, before integration of the putative transposable element . A second possibility is that these genes arose from rag - mediated dna rearrangement events that occurred in the germline, an operation that violates the precept that the rag recombinase is functional only in developing lymphocytes . A recent paper by lee et al . Addresses these questions by examining immunoglobulin genes in the nurse shark . The nurse shark ns4 immunoglobulin light chain gene family provided very useful information, as there are several highly homologous genes present both in pre - rearranged and unrearranged forms . These features allowed the authors to evaluate sequences in sufficient detail to ascertain whether the genes bear characteristic features of v(d)j recombination or footprints of transposition . Their analysis revealed that the pre - rearranged genes did indeed contain tell - tale signs of coding joints formed by v(d)j recombination, including both n nucleotides and p nucleotides, which strongly suggest a hairpin intermediate . The presence of these features in several junctions suggests several independent germline v(d)j recombination events, although analysis of multiple unrelated individuals suggests that these events are not frequent . Importantly, analysis of the unrearranged ns4 genes failed to detect the target site duplications that are hallmarks of transposon insertions . Thus, while the pre - rearranged ns4 genes appear to have been derived from unrearranged genes by germline v(d)j recombination, there is as yet no evidence to support the hypothesis that the unrearranged genes were derived from the pre - rearranged genes by insertion of a transposable element; this is discussed in a recent review by lewis and wu . The recent studies of the nurse shark ns4 genes strongly suggest that v(d)j recombination events can occur in the germline . Moreover, phylogenetic analysis indicates that these events occurred recently (at least from an evolutionary perspective), some time within the last 7 million years . What evolutionary benefit might there be in germline recombination events? Pre - rearranged immunoglobulin genes may confer certain advantages over genes that must be assembled by recombination in individual lymphocytes . For example, pre - rearranged genes may encode receptors capable of recognizing common pathogens likely to be encountered during the neonatal period, before the development of a full repertoire of rearranged antigen receptors . Furthermore, germline joining could have contributed to evolution of gene segment clusters, and possibly the evolution of d segments . To my knowledge, co - expression of both rag proteins outside the lymphoid system has not been reported, but rna species encoding rag-1 and rag-2 have been detected in zebrafish ovary and xenopus oocytes, respectively . Even if the recombinase is not normally expressed in these tissues, though, inappropriate expression might occur occasionally, perhaps as a result of improper reprogramming of tissue - specific gene expression during development . If rag expression does occur during the development of germ cells, another important question arises: how are loci chosen for rearrangement? During normal b - lymphocyte differentiation, immunoglobulin loci undergo a carefully orchestrated series of rearrangements . D to j rearrangements of the heavy - chain genes occur first, followed by v to dj rearrangements, followed in turn by rearrangement of the light - chain genes . The carefully ordered sequence of rearrangements is critical for proper lymphocyte differentiation and is thought to be controlled by accessibility of the loci, mediated by alterations in chromatin structure (reviewed in). Rsss present in antigen - receptor loci that have not been' targeted' for rearrangement are used rarely, if at all . The careful control of locus accessibility seen in lymphocyte differentiation may not be recapitulated in the development of germ cells - after all, these cells are not supposed to be expressing rag recombinase activity . Furthermore, the v(d)j recombinase is not particularly picky about the sequences it can target for rearrangement . Lewis and co - workers have found that' cryptic' sites capable of supporting rearrangement occur at least once in every 600 base pairs of a commonly used plasmid sequence, and they have estimated that there are at least 10 million such sites in the mammalian genome . In fact, there is evidence that cryptic sites in the mammalian genome can serve as targets for rag - mediated rearrangement in lymphocytes (reviewed in). Thus, it is possible that rag expression during germ - cell development might cause rearrangements of regions of the genome far removed from the immune receptor loci . These considerations suggest that the v(d)j recombinase might have been (and could still remain) a significant force shaping vertebrate evolution, by catalyzing v(d)j - like rearrangements and, perhaps, transposition . Comparison of genome sequences from a variety of organisms may allow some aspects of this notion to be tested . First, the rag proteins bind to the rsss (triangles) and bring them together into a synaptic complex . Cleavage ensues, generating a pair of blunt signal ends and a pair of dna hairpin coding ends . Joining of these ends generates signal and coding joints, respectively . A fragment of dna generated by rag - mediated cleavage, with rag proteins bound to the signal ends (the donor), can capture another dna duplex (the target). The rag proteins bound to the signal ends catalyze integration into the target, generating a characteristic duplication of the target sequence at the integration site (arrowheads). I thank vicky brandt for editorial assistance and mark landree for critical comments on the manuscript . Work in the author's laboratory is supported by the howard hughes medical institute and by grants from the national institutes of health, the human frontiers science program, and the american cancer society.
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Pharmaceutical companies and many academic centers have thousands of high - quality compounds that represent potential new drugs . These high - quality compound collections contain thoroughly studied development candidates and, in many cases, old drugs that were generated for non - cns disorders but that might be useful therapies for one or more psychiatric diseases . However, testing these compounds for activity relevant to treating psychiatric diseases is challenging . Whereas some disease areas, such as cancer or infectious disease, are relatively amenable to high - throughput in vitro testing of hundreds of thousands of compounds, psychiatric diseases are not . Because psychiatric diseases generally result from disorders of cell cell communication or circuitry, intact systems are required to detect improvement in disease - relevant endpoints . The need for observers to be blinded to the treatment group of each animal increases the resources required to appropriately dose and randomize animals in a way that is invisible to the observer . Furthermore, multiple complementary behavioral assays are required to determine whether observed effects are robust and are not due to undesired effects, such as sedation (crawley and paylor, 1997). Thus, the discovery of new neuropharmacological drugs with novel mechanisms of action is impeded by the absence of high - throughput, robust, and objective behavioral assays . Very recently a 96-well behavioral assay measuring the photomotor response of zebrafish embryos demonstrated that different classes of neuropharmacological compounds cause distinct patterns of responses (kokel et al ., 2010). Such an assay is relatively inexpensive and is well suited to screening high numbers of compounds about which little is known . For high - quality compounds, particularly compounds that have entered human testing, a rodent system offers advantages . For advanced compounds, rodent pharmacokinetics and safety are often known, and sufficient quantities of these compounds generally exist to support rodent studies . To enable testing of many compounds for behavioral effects relevant to psychiatric disease, psychogenics developed smartcube, an automated system in which behaviors of compound - treated mice are captured by digital video and analyzed with computer algorithms (brunner et al ., 2002). Pgi analytical systems uses data from smartcube to compare the behavioral signature of a test compound to a database of behavioral signatures obtained using a large set of diverse reference compounds . In this way, the neuropharmacological effects of a test compound can be predicted by similarity to major classes of compounds, such as antipsychotics, anxiolytics, and antidepressants . This approach is ideally suited to screening collections of existing drugs or drug candidates for previously unknown neuropharmacology, which could expedite the development of new and unexpected treatments for psychiatric disorders . In this paper, we demonstrate that this system can detect relevant cns activity of compounds representing mechanisms beyond those used clinically to treat psychiatric disorders . An overview of pgi analytical systems, a rapid, computer vision - enabled murine screening system for neuropharmacological activity, is shown in figure 1 . The pgi analytical systems training set consisted of hundreds of doses of clinically approved reference drugs, grouped per indication (e.g., anxiolytics, antidepressants, etc . ). Drugs were injected 15 min before the test, and multiple challenges were presented over the course of the test session . Digital videos of the subjects were processed with computer vision algorithms to extract over 2,000 dependent measures including frequency and duration of behavioral states such as grooming, rearing, etc ., and many other features obtained during the test session . Pgi analytical systems combine proprietary hardware, robotics, computer vision, and data mining techniques to collect thousands of behavioral features in a single test session and extract unique signatures for each compound . These signatures are compared with the signatures obtained from reference compounds (shown to have clinical efficacy) in different therapeutic classes and subclasses to predict clinical utility . A proprietary machine learning algorithm was developed to train a probabilistic classifier that mapped the extracted computer vision feature values from the training set to their corresponding cns indications . This tool was used to establish a reference database of therapeutic class signatures and provided a mechanism to determine the cns probabilistic profile of an arbitrary test sample . Our reference database comprises 14 classes of drugs with some of the major classes, such as the antidepressant class, comprising several subclasses with representatives of most of the drugs in the market . Test set assessment were chosen from our evaluation tests and two separate types of classifiers were built that make independent predictions: one at the therapeutic class level and one at the level of highly performing subclasses . The behavioral signatures of the test drugs were scored by these classifiers to predict potential therapeutic utility . To evaluate the ability of the pgi analytical system to detect relevant behavioral responses to novel compounds, we tested two compounds that represent mechanisms not yet used clinically to treat psychiatric disorders but that impact relevant behavioral endpoints in rodents . The two test compounds, tp-10 and pf-670462, had not been included in the computer algorithm training set . Tp-10 is a sub - nanomolar inhibitor of pde10a, a dual - substrate phosphodiesterase expressed in medium spiny neurons of the striatum that regulates striatal output by regulating both camp and cgmp hydrolysis (strick et al ., 2007). Tp-10 demonstrates multiple behavioral effects in rodents that are consistent with clinically effective antipsychotics, including decreased locomotor activity, inhibition of conditioned avoidance response, and improvement of amphetamine - induced deficits in auditory gating (schmidt et al ., 2008). Pf-670462 is a casein kinase i (ck1) inhibitor (badura et al ., 2007). All compounds were dissolved in a pharmasolve, peg, pg mixture, and were injected i.p . 13 h before the behavioral test because of its known effects on circadian rhythm at 50 mg / kg s.c . (badura et al ., 2007); this procedure was done both in the morning and evening . As a follow - up test to confirm the anxiolytic signature of pf-670462 we use the marble burying test . Pf-670462 was dissolved in 40% cyclodextrin and injected at 10 and 30 mg / kg, s.c . Experimenters were blind to the mechanisms of action of both compounds and to the dose being used . The institutional animal care and use committee of psychogenics reviewed and approved the animal use in these studies . The animal care and use program is fully accredited by the association for assessment and accreditation of laboratory animal care, international . In the pgi analytical system, tp-10 produced a dose - dependent signature of activity most similar to that of known antipsychotics, particularly at the 1-mg / kg dose (figure 2). Response curve in this system, consistent with its steep dose response curve in other rodent models (schmidt et al ., 2008). At the lower 0.5 mg / kg dose, tp-10 s activity is more similar to that of the vehicle than to any class of drugs in the reference database . At the higher dose of 2 mg / kg, tp-10 produced a behavioral pattern that is quite different from that of the vehicle yet not specific to any one class . Notably, tp-10 activity at the 2-mg / kg dose has a large behavioral component similar to that of high - dose antidepressants but not medium - dose antidepressants . Interestingly, three of the four reference antipsychotic signatures shown in figure 3 also have a larger high - dose antidepressant - like component at their highest doses . Overall, the profile of tp-10 in this computer - aided screening system is similar to that of clinically used antipsychotic drugs (gleason and shannon, 1997; powell et al ., 2008; this observation is consistent with tp-10 s activity in multiple rodent models (schmidt et al ., 2008), suggesting that the pgi analytical systems can identify antipsychotic - like activity of compounds acting through novel mechanisms . The pde10a inhibitor tp-10 produced a signature of activity suggesting potential antipsychotic activity . The size of a given colored region on each bar indicates the probability that the activity of tp-10 at that dose matches a given class of neuropharmacological agents . The size of the white area represents that probability that the activity is more similar to that of the vehicle rather than to that of any compounds in the reference database . Drug signatures of four of the antipsychotic drugs included in the reference dataset: aripiprazole, clozapine, haloperidol, and olanzapine . In contrast to pde10a, for which considerable literature data support its role as an antipsychotic target, the potential usefulness of altering ck1 s effects on circadian rhythm for treating psychiatric disorders is unknown . Ck1 is an important regulator of period proteins (per) that regulate the clock mechanism in cells (lee et al ., 2001). Clearly, improved regulation of circadian rhythm could improve functioning in patients with sleep disorders . Furthermore, disruptions of the sleep wake cycle can affect cognition, memory, and mood (benca et al . Ck1 is also involved in darp32 phosphorylation and thus participates in dopamine - dependent behavioral activation (bryant et al ., thus, the ck1 mechanism is a good candidate for screening in a system that may identify behavioral signatures relevant to a wide variety of psychiatric disorders . In the pgi analytical system at intermediate doses of 30 and 50 mg / kg, the ck1 inhibitor pf-670462 (badura et al ., 2007) demonstrated a signature consistent with anxiolytic, but not sedative / hypnotic, activity (figure 4). Because pf-670462 produces changes in circadian rhythm (badura et al ., 2007), we tested whether administering pf-670462 to mice 13 h prior to testing might produce a more relevant behavioral signature . Interestingly, the compound had almost no detectable behavioral effects in this system when dosed 13 h before testing, either in the morning (5:30 am) or the afternoon (7 pm). The anxiolytic signature of pf-670462 seen at the 30, but not 10, mg / kg dose was confirmed in the marble burying test, in which the higher dose decreased marbles buried without a decrease in locomotion (figure 5). The ck1 inhibitor pf-670462 demonstrated a signature consistent with anxiolytic activity (yellow) and, at the highest dose tested, behavioral effects similar to that of opiate analgesics (pink), although this dose is higher that doses tested previously in rodents and may reflect non - specific effects . 15 min before the test in studies represented by the five left - most bars of the chart . To assess potentially delayed behavioral effects due to its effects on circadian rhythm, pf-670462 was injected s.c . 13 h before testing in the morning (am) or in the afternoon (pm). The ck1 inhibitor pf-670462 shows anxiolytic activity in the marble burying test with a strong yet non - significant trend at 10 mg / kg and a significant effect at 30 mg / kg, consistent with the dose response anxiolytic signature uncovered by the high - throughput screen [anova f(4,43)> 8.8, p <0.0001; fisher plsd p <0.02]. The drug was injected s.c . Our work demonstrates the usefulness of a computer vision - based system for identifying behavioral effects of novel compounds and mechanisms in a relatively high - throughput in vivo assay . As one example, the pde10a inhibitor tp-10 demonstrated a behavioral signature in the pgi analytical system that was more similar to that of known antipsychotics than to that of other classes of neuropharmacological agents . Although the efficacy of pde10a inhibition in humans has not been tested, the activity of tp-10 here is consistent with effects of clinically used antipsychotics in multiple rat in vivo assays (schmidt et al ., 2008). Importantly, the steep dose response curve of tp-10 was also consistent between the computer vision - based system and traditional rodent assays . Interestingly, tp-10 was ineffective in two mouse pre - pulse inhibition models (schmidt et al ., 2008), although it showed an antipsychotic - like signature in mice in the present study . The computer vision - based system, which measures hundreds of small behavioral events, may reveal more relationships because of its ability to assess compound effects more broadly than a model designed to test one specific functional hypothesis . The tp-10 behavioral signature at the highest dose tested suggested a small degree of activity similar to that of high - dose antidepressants . This feature was observed with the higher doses of most of the four reference antipsychotics in figure 3, as well, suggesting that the similarity is due to side effects caused by high doses of all such compounds . The computer vision - based system also suggested new hypotheses regarding the behavioral effects of ck1 inhibition . We found that moderate doses of pf-670462 produced activity similar to that of anxiolytic agents, but not sedative / hypnotics . We followed up this result in the marble burying test, an assay predictive of anxiolytic therapeutic potential, and confirmed that a dose of 30 mg / kg has anxiolytic activity (figure 5). In summary, our data demonstrate that the pgi analytics computer vision - based system rapidly detects relevant behavioral effects of compounds acting through new mechanisms . Because the assay and data analysis are highly automated, use of the system eliminates the need for subjective human observation and evaluation of behavior, thus eliminating significant resource requirements along with much of the potential for bias inherent in subjective measurements . This approach is ideally suited to screening libraries of existing drugs or drug candidates for previously unknown neuropharmacology, which could expedite the development of new and unexpected treatments for psychiatric disorders.
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It exerts a decongestant effect on the swollen nasal mucosa of patients with chronic or acute rhinitis; however, it causes adverse effects, such as urinary retention in males, because of its systemic action on -adrenergic receptors.1 although pseudoephedrine generally has little effect on voiding in young patients, its actual effect on voiding function remains unclear . Urology clinics and emergency departments are sometimes required to deal with males who experience dysuria, incomplete voiding, or acute urinary retention after taking pseudoephedrine . Most of these patients are aged over 50 years, and some may have coexisting benign prostate hyperplasia, which they may not report to their doctor . Other patients may be relatively younger or may not report any history of voiding dysfunction . Although pseudoephedrine is labeled as requiring extra caution while administration in males with benign prostate hyperplasia,2,3 no other specific precautions regarding patient characteristics, such as age, are mentioned . Some studies have reported about the adverse effects of pseudoephedrine, such as urinary retention in children and adult patients.15 however, these studies have not provided any information regarding the age dependency of such adverse effects . To the best of our knowledge, no study has yet investigated the effect of pseudoephedrine on voiding function in asymptomatic males . In this study, we used the international prostate symptom score (ipss) questionnaire to evaluate the effects of pseudoephedrine on voiding function in asymptomatic males who received pseudoephedrine for nasal congestion treatment . The institutional review board of linkou chang gung memorial hospital approved this prospective study (registration number 104 - 4791b). Between may and august 2015, we recruited males with acute or chronic rhinitis who visited our outpatient department for evaluation . Written informed consent was obtained from all patients before enrollment in this study . Because all patients complained of nasal obstruction, we considered prescribing pseudoephedrine for symptom relief . We excluded women, males who self - reported voiding problems, males who were already receiving medications for voiding dysfunction, and males who were currently receiving any medication that could affect voiding function (eg, antihistamines that can inhibit the bladder contractility). We used the ipss questionnaire to evaluate voiding dysfunction in the patients . It contains the seven - item american urological association (aua) symptom index and one question on quality of life (qol), which is used to evaluate lower urinary tract symptoms (luts) in males, such as the benign prostate hyperplasia - related voiding problem . The seven - item aua symptom index, developed and psychometrically validated by an aua measurement committee in 1992, reliably assesses the severity of luts and is responsive to changes in the treatment.6 in 1993, the world health organization adopted the eight - item ipss, which uses the same seven questions assessing the luts severity as the aua symptom index, along with an eighth disease - specific qol question that assesses severity associated with luts.7 a study reported the reliability of this questionnaire for the evaluation of women with lower urinary tract dysfunction.8 the seven questions are divided into the following two parts: ipss voiding (ipss - v) scores and ipss storage (ipss - s) scores.9 the ipss - v scores consist of weak stream, intermittency, straining, and feeling of incomplete bladder emptying, whereas the ipss - s scores consist of frequency, urgency, and nocturia . Each scoring ranges from 1 to 5 for a total of maximum 35 points . The eighth question on qol the patients were asked to complete the ipss questionnaire before and 1 week after taking oral pseudoephedrine . The results of the questionnaire were analyzed as the ipss total (ipss - t), ipss - v, ipss - s, and qol due to urinary symptoms (qol - us). We defined an increased ipss - t score after receiving the medication as subclinical worsening of voiding function . All premedication parameters were analyzed using the independent t - test to ascertain significant predictors . We used the paired samples t - test to analyze premedication and postmedication parameters in the overall patients and in age - stratified groups (<50 and 50 years). The general additive model was used to examine the relationship between age and deteriorated voiding after medication . Most statistical analyses were performed on a personal computer by using the statistical package spss for windows (version 17.0) (spss inc ., chicago, il, usa) and the r software (version r 2.14.0; r & r, auckland, new zealand) for general additive models . The institutional review board of linkou chang gung memorial hospital approved this prospective study (registration number 104 - 4791b). Between may and august 2015, we recruited males with acute or chronic rhinitis who visited our outpatient department for evaluation . Written informed consent was obtained from all patients before enrollment in this study . Because all patients complained of nasal obstruction, we considered prescribing pseudoephedrine for symptom relief . We excluded women, males who self - reported voiding problems, males who were already receiving medications for voiding dysfunction, and males who were currently receiving any medication that could affect voiding function (eg, antihistamines that can inhibit the bladder contractility). It contains the seven - item american urological association (aua) symptom index and one question on quality of life (qol), which is used to evaluate lower urinary tract symptoms (luts) in males, such as the benign prostate hyperplasia - related voiding problem . The seven - item aua symptom index, developed and psychometrically validated by an aua measurement committee in 1992, reliably assesses the severity of luts and is responsive to changes in the treatment.6 in 1993, the world health organization adopted the eight - item ipss, which uses the same seven questions assessing the luts severity as the aua symptom index, along with an eighth disease - specific qol question that assesses severity associated with luts.7 a study reported the reliability of this questionnaire for the evaluation of women with lower urinary tract dysfunction.8 the seven questions are divided into the following two parts: ipss voiding (ipss - v) scores and ipss storage (ipss - s) scores.9 the ipss - v scores consist of weak stream, intermittency, straining, and feeling of incomplete bladder emptying, whereas the ipss - s scores consist of frequency, urgency, and nocturia . Each scoring ranges from 1 to 5 for a total of maximum 35 points . The eighth question on qol the patients were asked to complete the ipss questionnaire before and 1 week after taking oral pseudoephedrine . The results of the questionnaire were analyzed as the ipss total (ipss - t), ipss - v, ipss - s, and qol due to urinary symptoms (qol - us). We defined an increased ipss - t score after receiving the medication as subclinical worsening of voiding function . All premedication parameters were analyzed using the independent t - test to ascertain significant predictors . We used the paired samples t - test to analyze premedication and postmedication parameters in the overall patients and in age - stratified groups (<50 and 50 years). The general additive model was used to examine the relationship between age and deteriorated voiding after medication . Most statistical analyses were performed on a personal computer by using the statistical package spss for windows (version 17.0) (spss inc ., chicago, il, usa) and the r software (version r 2.14.0; r & r, auckland, new zealand) for general additive models . In total, 131 patients completed the questionnaire before and 1 week after receiving pseudoephedrine . The scores obtained in the ipss questionnaire before and after the medication are listed in table 2 . The ipss - t, ipss - v, and ipss - s scores slightly increased after the medication; however, the increase was not significant (ipss - t increased from 6.49 to 6.77, ipss - v from 3.33 to 3.53, and ipss - s from 3.17 to 3.24). The qol - us score nonsignificantly decreased from 2.02 to 1.87 . Among the 131 patients, 52 (39.7%) had lower ipss - t scores after the medication, whereas 37 (28.2%) and 42 (32.1%) had unchanged or higher scores . The numbers and percentages of the patients with changes (increased, unchanged, or decreased) in the ipss - v, ipss - s, and qol - us scores after the treatment with pseudoephedrine are listed in table 2 . We divided all the patients into two subgroups by defining an increased ipss - t score after pseudoephedrine treatment as the subclinical worsening of voiding function and an unchanged or decreased ipss - t score as unchanged voiding function . The independent t - test was used to analyze differences in age and premedication ipss - t, ipss - v, ipss - s, and qol - us scores between these two subgroups . We observed that age and premedication ipss - v score significantly differed between the subclinical voiding dysfunction and unchanged voiding function subgroups (p<0.05). The mean age and premedication ipss - v score in the subclinical voiding dysfunction subgroup were 49.6 years and 4.57, respectively, and those in the unchanged voiding function subgroup were 38.4 years and 2.74, respectively . The ipss - t, ipss - v, ipss - s, and qol - us scores did not significantly differ before and after the medication in the overall patients . However, the scores differed after stratifying the patients according to their age, those 50 and <50 years . In the 50-year group, the ipss - t, ipss - v, and ipss - s scores significantly increased after the medication (ipss - t increased from 9.95 to 11.45, ipss - v from 5.38 to 6.07, and ipss - s from 4.57 to 5.38), whereas the qol - us score nonsignificantly decreased from 2.71 to 2.48 (p=0.057). In the <50-year group, all the scores decreased but not significantly . The difference in the symptom score change between the two age - stratified groups is presented in figure 1 . Of the 131 patients, the ipss - t score after the medication increased in 42 patients (32.1%). After the treatment, 51.8% of the 56 patients in the 50-year group and only 17.3% of the 75 patients in the <50-year group experienced subclinical voiding dysfunction . The general additive model plots revealed the changes associated with deteriorated voiding function in different age groups (figure 2). Most patients younger than 52.8 years exhibited no change or slight improvement in voiding function after the medication . By contrast, most patients older than 52.8 years exhibited deterioration in voiding function after the medication, and the difference increased with age . Throughout the study period, three of the 131 patients, aged 29, 51, and 62 years, respectively, experienced symptomatic dysuria and visited urological clinics . Its principal mechanism of action relies on its direct effect on the adrenergic receptor system.10,11 the vasoconstriction produced by pseudoephedrine is believed to be principally an -adrenergic - receptor response.12 the -adrenergic receptors are located on muscles lining the walls of blood vessels, and when these receptors are activated, the muscles contract, causing the constriction of blood vessels . In addition, pseudoephedrine acts on 2-adrenergic receptors to induce relaxation of smooth muscles in the bronchi.10,11 along with its therapeutic effect on nasal congestion, pseudoephedrine produces adverse effects, such as restlessness, nervousness, dizziness, stomach pain, vomiting, weakness, hallucination, palpitation, difficulty in breathing, and urinary retention . The main cause of urinary retention or dysuria after the pseudoephedrine treatment is the effect of pseudoephedrine on 1a- and -adrenoceptors . The 1a - subtype is highly expressed and promotes contraction of the bladder neck, urethra, and prostate to enhance the bladder outlet resistance, particularly in older males with enlarged prostates . The -subtype mediates the relaxation of smooth muscles in the bladder.13 the main effect of pseudoephedrine on the lower urinary tract tends to maintain a continent bladder . Pseudoephedrine is effective in the treatment of certain forms of incontinence.14 in addition, an animal study reported clinical improvement in female incontinent dogs after pseudoephedrine treatment, despite the lack of statistically significant changes in urodynamic variables.15 by contrast, pseudoephedrine failed to treat the incontinent bladder in primary nocturnal enuresis in children.16 in 1928, an adult patient was first reported to suffer from voiding difficulty during pseudoephedrine treatment in boston,4 which was followed with reports on the first pediatric cases of urinary retention after pseudoephedrine treatment in 1977.1,17 since the first case, pseudoephedrine - related dysuria or urinary retention has occasionally been reported in urological or emergency departments . Urinary retention can be detected at lower doses of pseudoephedrine; however, few cases have been reported.18 therefore, although pseudoephedrine is contraindicated for patients with urinary retention and should be administered with caution in patients with bladder outlet obstruction,2,17 it is still prescribed as a safe decongestant for patients without voiding problems . In this study, we investigated changes in voiding function after pseudoephedrine treatment in males without coexisting voiding dysfunction . In our study, only 2.3% of the patients experienced symptomatic dysuria and required further management after receiving pseudoephedrine . However, among the patients who did not self - report voiding dysfunction, ~30% had increased ipss - t scores, defined here as subclinical voiding dysfunction . Voiding dysfunction is significantly correlated with age in males, and the prostate enlargement has a crucial role.18 thus, we divided our patients into age - stratified groups . The percentage of voiding dysfunction increased with age and increased to more than 50% (51.8%) in patients aged> 50 years; it was only 17.3% in patients aged <50 years . This result suggested that older age is a potential risk factor for pseudoephedrine - related dysuria or urinary retention . By dividing the patients into subclinical voiding dysfunction and unchanged voiding dysfunction subgroups, we observed that older patients and those with higher pre - medication ipss - v scores (poor baseline voiding function) were more likely to have subclinical voiding deterioration . Such results imply that apart from older age, patients with undiagnosed lower urinary tract disease may develop sub - clinical voiding dysfunction and even clinical dysuria during pseudoephedrine treatment . The ipss - t score consists of ipss - v and ipss - s scores; the former represents the voiding function and the latter represents the storage function . Pseudoephedrine causes contraction of the bladder neck, urethra, and prostate to enhance the bladder outlet resistance, which might impair patients voiding ability . This is consistent with our result that compared with a higher ipss - s score, a higher ipss - v score is a more favorable risk predictor of subclinical voiding dysfunction in patients receiving pseudoephedrine . Our study has some limitations, which are as follows: 1) we analyzed only males in this study; thus, whether pseudoephedrine exerts a similar effect in women requires further evaluation; 2) subclinical voiding dysfunction does not indicate a need for further medical assistance; therefore, examination of the number of patients requiring assistance and relevant associations will require a large sample size; 3) we recruited patients who received oral pseudoephedrine at a standard dosage for 1 week; thus, whether the outcomes differ at varying doses or durations remains unclear; and 4) the ipss questionnaire only represents the subjective feeling of voiding problem; therefore, objective examinations, such as uroflowmetry and urodynamic study, are required to investigate the actual effect of pseudoephedrine on patients voiding function . Although pseudoephedrine is often relatively safe in younger males and seldom requires further medical assistance, it might result in subclinical voiding dysfunction in older males . Treatment with pseudoephedrine requires extra precautions in males aged> 50 years and in those with coexisting voiding symptoms.
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This figure is expected to grow by 20% over the next decade as the aging population increases and lives longer . This disease group is the fourth biggest killer in the developed world after heart diseases, cancer, and stroke . The most common neurodegenerative diseases are ad, parkinson disease, lewy body dementia, frontotemporal dementia, and amyotrophic lateral sclerosis . The most widely recognized is ad, which is among the principal debilitating conditions of the current century . Approximately 24 million people worldwide suffer from dementia, 60% of cases being due to ad, which occurs in 1% of individuals aged 50 to 70 years old and dramatically increases to 50% of those over 70 years old . Dramatically, these numbers are estimated to increase to 15 million in the next 40 years . From the neuropathological point of view, ad is characterized by selective neuronal loss, marked synaptic alteration, morphological mitochondrial abnormalities, and tau pathology . The extracellular amyloid plaques mainly composed of amyloid - beta and intracellular neurofibrillary tangles built up of hyperphosphorylated tau, although the molecular mechanisms underlying the disease are still unknown and there is still no cure . Many lines of evidence suggest that oxidative stress is one of the earliest changes and plays an important role in the pathological process in ad, and more recently, energy deficiency and mitochondrial dysfunction have been recognized as a prominent, early event in ad [111]. Oxidative stress plays a critical role in the pathogenesis of ad and is intimately linked to aging, the best established risk factor for ad . Increased oxidative stress levels have been found in the brains of patients with ad in sweden and early in tg2576 app transgenic mice [12, 13]. Recently, we described that pla2g3 gene silencing produced a marked inhibition of the free radical - generating xanthine / xanthine oxidase- (x - xod-) system - induced cell death, and that pla2g3 polymorphisms are associated with ad in a spanish case - control sample . In previous studies with choroid plexus homogenates with different cases with ad in the different stages (i/0, iii - iv, and v - vi), we demonstrated that in ad patients, amyloid - beta peptide also accumulates in choroid plexus, there is an oxidation of carboxymethyl - lysine (cml), and n - carboxyethyl - lysine (cel) may result in impaired protein interactions, protein folding, and protein kinase activity; abnormal function of endothelial and vascular smooth muscle cells; impaired hdl - cholesterol metabolism in the choroid plexus in advanced stages of ad . Mitochondria are found in virtually all eukaryotic cells and function to generate cellular energy in the form of adenosine triphosphate (atp) by oxidative phosphorylation and are thought to be derived evolutionarily from the fusion of prokaryotic and eukaryotic organisms . They are also involved in regulation of cell death via apoptosis, in the control of cell division and growth, in calcium homeostasis, haem biosynthesis, and in the formation and export of iron - sulphur clusters . Mitochondria are composed of a double lipid membrane which structures four compartments, distinct by composition and function (figure 1). It contains many proteins like import complexes and voltage - dependent anion channels responsible for the free passage of low - molecular - weight substances (up to 5000 da) between the cytoplasm and the intermembrane space which represents a reservoir of protons establishing a proton electrochemical gradient across inner mitochondrial membrane that is needed for the production of atp via atpase (complex v). Intermembrane space contains proapoptotic proteins like cytochrome c, smac / diablo, endog, and htra2/omi . In contrast to the permeable outer membrane, the inner mitochondrial membrane, rich in cardiolipin, provides a highly efficient barrier to the flow of small molecules and ions, including protons . It houses the respiratory enzymes of the electron transport chain, the cofactor coenzyme q, and many mitochondrial carriers . In the matrix, mitochondria are unique amongst cellular organelles in that they have their own, circular, double - stranded dna (mtdna) which is inherited almost exclusively down the maternal line and codes for 37 mitochondrial genes, 13 of which translate to proteins involved in oxidative phosphorylation [18, 19]. The remaining genes encode transfer (22 genes) and ribosomal (2 genes) rna allowing the mitochondria to generate their own proteins . Although mitochondria have the ability to produce proteins, the vast majority of proteins that function within the mitochondria, including those involved in dna transcription, translation, and repair, are encoded by nuclear dna and are transported into the mitochondria from the cytosol . As mtdna is located in the mitochondria in close proximity to the electron transport chain, it is more susceptible to damage from free radicals generated during oxidative phosphorylation . Mitochondria generate energy by two closely coordinated metabolic processes: krebs cycle and the oxidative phosphorylation (oxphos). Oxphos is made up of the electron transport chain assembled in four enzymes (complex i to iv) as well as the f1f0-atp synthase (complex v). Complexes i, iii, and iv are located in inner mitochondrial membrane as integral proteins, whereas complex ii is attached to the inner surface of this membrane . The function of the chain is to generate cellular energy in the form of atp . These five enzymes of the complex are connected functionally by mobile electron acceptors and donors: ubiquinone and cytochrome c. electrons from nadh and fadh2 are fed into complexes i and ii, respectively . Ubiquinone q carries electrons from both complexes to complex ii, and cytochrome c does it from complex iii to iv reducing molecular oxygen to water . As electrons are transferred along electron transport chain, a fixed number of protons are pumped from the matrix into inner membrane space establishing a electrochemical gradient characterized with a specific electrical potential . The redox energy drives the synthesis of atp from adp as protons are transported back from inner membrane space into the matrix via complex v. accumulation of amyloid - beta in ad brains is thought to underlie neuronal dysfunction and memory loss, being centrally implicated in ad pathogenesis . Moreover, previous to mitochondrial accumulation of amyloid - beta showed in ad patient and ad transgenic mouse brain, the toxic amyloid - beta species has to be accumulated in the cytosol of the cells . Many studies showed amyloid - beta interaction with different receptors in the cellular membrane of the vasculature, neurons, oligodendrocytes, and glial cells where it is transported from cell surface into endosomal and lysosomal compartments [2123]. The aberrant signalling of these receptors in ad triggered an abnormal accumulation of amyloid - beta into cytosol - inducing cellular stress underlies to neuronal dysfunction and dementia . It is described by our group and others that these receptors can be megalin, also known as low - density lipoprotein - related protein-2 (lrp2), lrp-1, or rage (receptors for advanced glycation end products). The interaction of these receptors with amyloid - beta in neurons, microglia, and vascular cells accelerates and amplifies deleterious effects on neuronal and synaptic functions . These findings are further in line with the recently proposed hypothesis of an intracellular amyloid - beta toxicity cascade which suggests that the toxic amyloid - beta species intervening in molecular and biochemical abnormalities may be intracellular soluble aggregates instead of extracellular, insoluble plaques . There are many studies proposing that megalin- and/or rage - dependent signalling are involved in the regulation of amyloid - beta clearance and probably may contribute to amyloid pathology and cognitive dysfunction observed in the ad patients and ad mouse model . Studies of postmortem brains from ad patients and transgenic mouse models of ad suggest that oxidative damage, induced by amyloid - beta, is associated with mitochondria early in ad progression . Amyloid - beta and app protein are known to localize to mitochondrial membranes, block the transport of nuclear - encoded mitochondrial proteins to mitochondria, interact with mitochondrial proteins, disrupt the electron transport chain, increase reactive oxygen species (ros) production, cause mitochondrial damage, and prevent neurons from functioning normally . Recent scientific research has identified multiple mechanisms of amyloid - beta interaction with mitochondria at different mitochondrial compartments: the outer mitochondrial membrane, intermembrane space, inner mitochondrial membrane, and the matrix . It is well known that the involvement of amyloid - beta - induced mitochondrial dysfunction in ad pathogenesis, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the alzheimer mitochondrial cascade hypothesis . The brain is vulnerable to oxidative stress owing to its high lipid content, its relatively high oxygen metabolism, and its low levels of antioxidant defenses . One of the most interesting events in ad is that mitochondrial oxidative stress occurs early in ad progression, before the onset of amyloid - beta pathology [27, 28]. Oxidative stress was also reported in the mitochondria of other tissues different to the brain such as platelets and fibroblasts from ad patients [29, 30]. Free radicals (compounds with an unpaired electron) or ros are a normal part of metabolism . Mitochondria are the major source of ros, and, in fact, mitochondrial dysfunction as well as hypometabolism has long been implicated in the onset of the familial and sporadic forms of ad . Quantitative morphometric, molecular, and cellular analysis of mitochondria shows increased abnormal and damaged mitochondria in ad [33, 34]. Energy deficiency and mitochondrial dysfunction have been recognized as a prominent, early event in ad . Mitochondrial abnormalities have been found both in neurons and astrocytes [3537], suggesting that both neurons and astrocytes might be damaged by free radicals in the ad brain . Superoxide radicals might be produced in mitochondrial electron transport chain complexes i and iii and in components of the krebs cycle, including a - ketoglutarate dehydrogenase . H2o2 and superoxide radicals, released from the mitochondrial matrix and from the inner and outer mitochondrial membranes, might be carried to the cytoplasm and, ultimately, might lead to the oxidation of cytoplasmic proteins . Several lines of evidence suggest that app and amyloid - beta are factors contributing to mitochondrial dysfunction in ad (figure 2). Mitochondria were found to be the target both for amyloid precursor protein (app) that accumulates in the mitochondrial import channels and for amyloid - beta that interacts with several proteins inside mitochondria and leads to mitochondrial dysfunction . Multiple lines of evidence support app and amyloid - beta as contributing factors to mitochondrial dysfunction in ad: both app and amyloid - beta are present in mitochondrial membrane and interact with mitochondrial proteins, block mitochondrial import channels, impair mitochondrial transport, disrupt the electron transfer chain, increase ros levels, and cause mitochondrial damage . With regard to localization of app in mitochondria, it was demonstrated that app formed stable 480 kda complexes with the translocase of the outer mitochondrial membrane 40 (tom40) import channel and a supercomplex of approximately 620 kda with both mitochondrial tom40 and the translocase of the inner mitochondrial membrane 23 (tim23) import channel tim23 in an n (in mitochondria) -c (out of cytoplasm) orientation . Interestingly, in brain tissues of ad - affected subjects, app localized with mitochondria fraction, associated to tom40 and tim23, in a translocation - arrested manner, that may prevent import of de novo synthesised nuclear - encoded mitochondrial protein, such as subunits of the electron transport chain . In agreement with the intracellular localization of app, cell studies showed mitochondrial accumulation of amyloid - beta in ad patients and app mouse transgenic mouse brain . In transgenic app mice, mitochondrial amyloid - beta accumulation increased at around 4 months of age, well before the formation of plaques . In total, these findings are further in line with the recently proposed hypothesis of an intracellular amyloid - beta toxicity cascade which suggests that the toxic amyloid - beta species intervening in molecular and biochemical abnormalities may be intracellular soluble aggregates instead of extracellular, insoluble plaques . App and amyloid - beta may block mitochondrial translocation of nuclear - encoded proteins, such as components of the electron transport chain [4345], impairing mitochondrial function . Intramitochondrial amyloid - beta is able to perturb mitochondrial function in several ways by directly influencing extracellular transport chain complex activities, impairing mitochondrial dynamics, or disturbing calcium storage [47, 48], thus increasing apoptotic pathways . Moreover, amyloid - beta interacts with mitochondrial matrix components inducing an improper mitochondrial complex function leads to a decreased mitochondrial membrane potential of the organelle and impairing atp formation . In app processing, oligomeric amyloid - beta is hypothesized to enter in the mitochondria by penetrating the membrane because amyloid - beta is enriched at synaptic terminals . In support of the hypothesis that app and amyloid - beta enter mitochondria, several studies have found app and its derivatives (monomeric and oligomeric forms of amyloid - beta) in mitochondrial membranes [53, 54]. Amyloid - beta normally interact with the mitochondrial matrix protein, amyloid - beta - binding alcohol dehydrogenase (abad), leading to mitochondrial dysfunction . Caspersen et al . Showed amyloid - beta in mitochondria from postmortem brain specimens of ad patients and an accumulation of amyloid - beta in the brain mitochondria from app mice . With digitonin fractionation analysis of isolated mitochondria from app mice revealed amyloid - beta in outer and inner mitochondrial membranes and matrix and that mitochondrial amyloid - beta decreases cytochrome oxidase activity and increases free radical production and carbonyl proteins . Further, in the amyloid interactions in mitochondrial proteins, two more studies found that app interacts directly with mitochondrial proteins . It was demonstrated that mitochondrial atp synthase subunit is a binding partner of the extracellular domain of app and amyloid - beta . Transfection of app - deficient neuroblastoma cells with app resulted in increased surface localization of the atp synthase a - subunit and in extracellular app and amyloid - beta inhibiting the extracellular generation of atp . In another study, the authors found that nonglycosylated full - length and c - terminal - truncated app accumulates in the protein import channels of mitochondria of human ad brains but not in age - matched controls . The accumulation of app across mitochondrial import channels inhibited the entry of nuclear - encoded cytochrome c - oxidase subunits iv and vb proteins and was associated with decreased cytochrome oxidase and increased free radical production . It is well documented that mtdna changes are responsible for aging phenotypes [5557]. For example, many tissues from aged individuals have a lower respiratory function compared with those from younger individuals . It has been hypothesized that ongoing oxidative damage to mtdna may be the underlying mechanism for cellular senescence . Since mtdna repair mechanisms are limited and because mtdna is situated in close proximity to the site of ros production, mtdna is more vulnerable to oxidative damage than nuclear dna . With age, oxidation of mtdna increases compared to nuclear dna leading to an age - dependent accumulation of mtdna mutations . Point mutations and deletions in mtdna are highly prevalent in aged cells, and there is evidence that 8-hydroxy-2- deoxyguanosine (damaged dna) is more prevalent in aged tissues . Further, mice carrying an mtdna mutation (in the dna polymerase - g gene) showed features of aging and reduced lifespan, suggesting that mtdna changes are crucial for aging phenotypes . Defects in mtdna have not only been found in elderly persons without ad but also in ad patients [61, 62] and have been associated with decreased cytochrome oxidase activity in non - ad aging and aging ad brains . One recent study found that somatic mtdna control region mutations are elevated in ad patients . These mutations would lead to an overall reduction in mtdna copy number which would result in a decrease in oxidative phosphorylation . This mutation inhibits complex i respiration which leads to increased ros production, decreased membrane potential, and subsequent calcium deregulation . The effects of these mutations may lead to opening of the mitochondrial permeability transition pore and subsequent neuronal death . Increased ros levels act at multiple levels to impair mitochondrial function: they induce mtdna mutations that consequently negatively influence mitochondrial function, enhance amyloid - beta production by guiding app cleavage pathway toward the amyloidogenesis, increase lipid peroxidation [67, 68], activate mitophagy, leading to a reduced mitochondrial number, and augment tau hyperphosphorylation and nft formation impairing organelle trafficking and neuronal function finally leading to apoptosis . Using quantitative rt - pcr techniques, it was measured mrna expression of 11 mitochondrial - encoded genes in patients with early ad and with definite ad, as well as in age - matched control subjects . This interesting analysis revealed a downregulation of mitochondrial genes in complex i of oxphos (oxidative phosphorylation) in brain of ad patients . Complex i showed a down regulation of mitochondrial genes, whereas complexes iii and iv showed increased mrna expressions in these ad brains, suggesting a great demand on energy production . In a previous paragraph, we reported a decrease of cytochrome oxidase in the mitochondria from platelets, fibroblasts, and brains of ad patients . To compensate for the loss of cytochrome oxidase, mitochondrial - encoded genes might be activated in the surviving brain neurons of ad, for those patients this chain of events has been interpreted as a compensatory response . Synaptic degeneration is an early pathological feature in ad and is closely correlated to impaired cognitive function and memory loss . Recent studies suggest that involvement of amyloid - beta peptide in synaptic mitochondrial alteration underlies these synaptic lesions . Based on recent findings in human ad subjects, ad animal models, and ad cellular models, synaptic mitochondria undergo multiple malfunctions including amyloid - beta accumulation, increased oxidative stress, decreased respiration, and compromised calcium handling capacity, all of which occur earlier than changes seen in nonsynaptic mitochondria prior to predominant ad pathology . Of note, the impact of amyloid - beta on mitochondrial motility and dynamics exacerbates synaptic mitochondrial alterations . Mitochondrial number is indeed very high in neurons, and mitochondria are especially enriched in synapses . Due to the limited glycolytic capacity of neurons thus, the importance of synaptic mitochondria in supporting synapses and the high vulnerability of synaptic mitochondria to amyloid - beta make them a promising target of new therapeutic strategy for ad . Synaptic mitochondria are synthesized in neuronal soma; they are then transported to dendrites and axones, are distributed abundantly around synapses where mitochondria modulate calcium balance, and actively provide energy to fuel the synaptic function [69, 70]. If mitochondria localized in the cell body are damaged or are otherwise degraded, such as by aging or by amyloid - beta, these defective mitochondria might be transported to synaptic terminals by natural mitochondrial trafficking, where they produce low levels of atp owing to their degradation . . Defects in synaptic mitochondria obviously compromise synaptic function being very vulnerable to accumulative damages . Found amyloid - beta oligomers in synaptosomal mitochondrial fractions and decreased energy metabolism in ad transgenic mice . Abnormalities of mitochondrial function, including decreased mitochondrial respiration, ros generation, and hypometabolism, occur in the ad brain [39, 72] and in brains of ad mouse models [73, 74]. Amyloid - beta accumulation in the synapses directly disturbs mitochondrial function, causing oxidative stress, decreased atp, and increased ca influx [9, 75]. Furthermore, the interaction of mitochondrial amyloid - beta with its binding proteins, such as abad and cypd [9, 75], exacerbates amyloid - beta - induced mitochondria and neuronal stress and malfunction . Further, it was recently showed that amyloid - beta impaired synaptic mitochondrial distribution, axonal mitochondrial mobility, and increased axonal mitochondrial fragmentation . These promising findings are in agreement with those of recent studies indicating that amyloid - beta causes rapid and severe impairment of mitochondrial transport and alters mitochondrial dynamics . Synaptic terminals are sites of high energy demand and require high levels of cellular atp for neurotransmitter exocytosis and the potentiation of neurotransmitter release . These studies suggest that in an amyloid - beta - rich environment, overt mitochondrial dysfunction occurs and that mitochondria provide a direct site for amyloid - beta - mediated cellular perturbation, causing synaptic mitochondrial dysfunction in ad . However, it is not yet known whether amyloid - beta accumulates predominantly in synaptic mitochondria and whether synaptic mitochondria enriched for amyloid - beta are more vulnerable, although it is well known that the increase in oxidative damage exhibited by synaptic mitochondria might affect neurotransmission and synaptic damage and loss and might be ultimately responsible for cognitive decline in ad patients . In view of the critical role of synaptic mitochondria in energy production, synaptic calcium buffering, and regulation of synaptic function, impaired movement to and sequestration of mitochondria at synapses by amyloid - beta could be a mechanism of the pathogenesis at the distal synapses, together with early changes in synaptic mitochondrial function . In ad is very well documented the intracellular deposits of amyloid - beta in the brain parenchyma; however, amyloid - beta also accumulates in choroid plexus epithelial cells and in cerebrovascular walls, where it induces blood - brain barrier disruption . Several studies have shown that amyloid - beta alters transmembrane and cytoplasmic tight junction proteins in brain microvessel endothelial cells and choroid plexus, which ultimately leads to disruption in the integrity of the blood - brain barrier [79, 80]. Abnormal patterns of mitochondrial stress protein expression are found in the choroid plexus and bbb of ad subjects . There was a trend towards increased expression of hsp60, a mitochondrial stress protein, compatible with mitochondrial pathology recently documented in choroid plexus of ad . It was recently described in choroid plexus homogenates from 27 cases with ad - related pathology in different stages (stages i/0, iii - iv/0-b and v - vi / b - c) increased carboxyethyl - lysine (cel) and carboxymethyl - lysine (cml) expression in ad cases stages ivb and v - vi / b - c when compared to controls and cases with ad - related pathology . Interestingly, the authors suggest that other factors in addition to local fibrillar amyloid - beta were associated with oxidative damage in the choroid plexus . Though two - dimensional gel electrophoresis in combination with mass spectrometry identified other proteins as targets of increased oxidative damage in ad (tyrosine 3/tryptophan 5-monooxygenase activation protein, zeta polypeptide, tropomyosin 3 isoform, and apolipoprotein a - ii). Recent results from our laboratory have suggested direct relationship between amyloid - beta accumulation at the choroid plexus epithelium and the development of functional and structural dysfunctions [77, 83]. In addition, we demonstrated the existence of a link between amyloid - beta - induced choroid plexus cell death, increased production of nitric oxide (no), and mitochondrial dysfunction in the choroid plexus of patients with ad and app / ps1 mice . In ad patients and app / ps mice, we found an alteration induced by amyloid - beta of the enzyme activity of the respiratory chain complex iv in the choroid plexus epithelial cells . Accumulation of amyloid - beta peptides is a critical event in the pathology of ad, because they induce multiple neurotoxic effects, including mitochondrial dysfunction . Based on these results, we considered a reduction of amyloid - beta with gelsolin such as primary therapeutic target . Gelsolin is an amyloid - beta binding protein that inhibits apoptosis, although the underlying mechanism is unclear . We observed that gelsolin reduces brain amyloid - beta burden in the app / ps1 mice, accompanied by an inhibition of nitric - oxide production and cell death, not only in the choroid plexus but also in the cerebral cortex . Gelsolin levels restored the impaired mitochondrial activity, resulting in the increase of cytochrome c - oxidase activity . Additionally, these result demonstrated that gelsolin plays an important role in decreasing amyloid - beta - induced cytotoxicity by inhibiting nitric oxide production and apoptotic mitochondrial changes . All these promising findings make gelsolin an appealing tool for the prophylactic treatment of ad . Based on findings from in vitro and in vivo studies, it has been proposed that amyloid - beta has a significant role in synaptic dysfunction and cognitive decline in ad patients . Among other facts, amyloid - beta accumulates at synaptic terminals and enters the mitochondria, especially in the synaptic mitochondria . Mitochondria have been implicated in the neurodegenerative diseases and the onset and progression of age - associated diseases since decades . The accumulation of mtdna changes might increase ros production and reduce mitochondrial atp in an age - dependent manner . Recent studies of neurons from postmortem ad brain specimens and from transgenic ad mouse brain specimens suggest that oxidative damage induces amyloid - beta production . Studies of postmortem brains from ad patients and transgenic mouse models of ad suggest that oxidative damage, induced by amyloid - beta, is associated with mitochondria early in ad progression . Amyloid - beta present within mitochondria may provide a direct link between mitochondrial dysfunction in ad and pathogenic amyloid - beta . Amyloid - beta associated with mitochondria may be deposited at several locations, and yet nobody has known if amyloid - beta enters only in presynaptic mitochondria, postsynaptic mitochondria, or both . Interestingly, amyloid - beta is not present exclusively on the outer mitochondrial membrane and also might be present at that site to influence the interaction of multiple cytosolic proteins (including those of the bcl2 family) with mitochondria, as well as affecting the receptor binding of cargo targeted for import into the organelle . Amyloid - beta and app are reported to localize to mitochondrial membranes, block the transport of nuclear - encoded mitochondrial proteins to mitochondria, interact with mitochondrial proteins, disrupt electron transport chain activities, increase ros production, cause mitochondrial damage, and prevent neurons from functioning normally . The mechanisms of amyloid - beta and app transport into mitochondrial membranes are unclear . Amyloid - beta accumulates at synaptic terminals and impairs synaptic function and also enters synaptic mitochondria and causes damage . Mitochondrial damage is expected to be greater in synaptic mitochondria than in cell body mitochondria . The damaged, synaptic mitochondria might not satisfy the high energy demands required at synapses, which might lead to impaired neurotransmission and, ultimately, to cognitive failure . Based on these concepts, therapies targeting basic mitochondrial processes, such as energy metabolism or free radical generation, or specific interactions of disease - related proteins with mitochondria, hold great promise and future . In ad, tremendous progress has been made in developing therapeutic strategies to decrease amyloid - beta production and toxicity . However, further research is needed to develop molecules that target intact amyloid - beta in brain neurons affected by ad . Further research is also needed to test the efficacies of mitochondrially targeted antioxidants in ad mouse models . Future experiments that focus on the functional association of mitochondria with app and amyloid - beta might be useful for identifying mitochondrial drug targets.
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Although blood oxygenation level - dependent (bold) functional mri has assumed a role of great importance in systems neuroscience, our understanding of factors determining the amplitude and spatial extent of the bold effect under different conditions remains incomplete . Relevant parameters include baseline values and reactive capacity for cerebral perfusion, oxidative metabolism, and blood volume . An understanding of how these contribute to the bold response is important, since in general they may vary due to age or disease, and also depending on the nature of the neural system targeted by an applied stimulus . In particular, the exact nature and extent of the coupling between changes in oxidative metabolism and perfusion increases during neuronal activation is still the subject of debate . While recent studies have focused on quantification of responses during nonspecific activation of diffuse regions of sensory and motor cortex [14], this topic has also arisen in the context of highly localized responses in cortical columnar systems [5, 6]. In the present study, we sought to bridge the gap between these two regimes by looking at the effect of selective activation of only part of a small - scale cortical columnar system on the apparent bold response observed at a spatial resolution typical of studies used in human subjects . Early optical imaging studies [79] suggested that although evoked changes in oxidative metabolism exhibit a high degree of spatial specificity, brain perfusion is regulated on a much coarser spatial scale . If this is true, then there might be profound implications for the bold mri signal, especially when measured during manipulations such as monocular stimulation, which preferentially activates the set of ocular dominance columns projecting to the stimulated eye . In particular, one might expect the spatial pattern of perfusion response evoked by stimulation of a single eye to be similar to that seen during binocular stimulation, despite a substantial reduction in the metabolic response (compared to binocular stimulation) in columns projecting to the occluded eye . Since the bold signal reflects changes in the level of venous deoxygenated hemoglobin, this gratuitous hyperperfusion in unstimulated ocular dominance columns could be expected to result in a higher bold signal at a given level of perfusion increase (considering spatial averages over multiple columns, which would be applicable at commonly used spatial resolutions in fmri). The present study examines joint changes in perfusion and bold signals during monocular and binocular stimulation, to test the hypothesis that spatial decoupling of flow and metabolic responses during stimulation of only a partial subset of the columnar regions distributed within primary visual cortex leads to a significant shift in the ratio between spatially averaged bold and perfusion signals (measured using arterial spin - labeling). By combining quantitative mri - based measures of these two physiological quantities, we hope to provide new insight into the spatial precision with which cerebral blood flow is regulated, as well as factors which determine bold contrast amplitude in cortical tissues exhibiting columnar organization . Eight healthy subjects (five males and three females) 24 2.6 years old, one left eye and hand dominant (male) and seven right eye and hand dominant, participated in the study . The subjects did not suffer from any known visual deficits except myopia (mri - compatible corrective glasses were fitted in these cases). All gave informed consent and the project was approved by the comit mixte dthique de la recherche du regroupement neuroimagerie / qubec . Data from two of the subjects was not analyzed due to the poor quality of the arterial spin - labeling (perfusion) data . Subjects were fitted with a neoprene rubber mask which allowed occlusion of one eye by a removable patch . The patch was applied and removed as needed between the appropriate scans, by an experimenter, from the back of the scanner bore . Each scanning session included eight six - minute acquisitions, during which alternating one - minute blocks of baseline (uniform grey screen with central fixation point) and one - minute blocks of visual stimulation (black and white checkerboard reversing contrast at a rate producing four white periods per second within a square) were presented, starting with baseline . During each scanning run, the subject received either binocular (b) or monocular (m) stimulation to their nondominant eye with separate scans conducted in the following order: b - b - m - m - b - b - m - m . Subjects were instructed to direct their gaze at the central fixation point throughout all scans . The nondominant eye was selected for monocular stimulation to maximize the difference in activation between the monocular and binocular trials given that there may presumably be more extensive activation of v1 for the dominant eye . Mri data acquisition was carried out using a siemens trio 3 tesla mri system, at software revision va25a . Images reflecting relative perfusion were acquired using a picore / q2tips arterial spin - labeling (asl) acquisition [11, 12]. The spatial resolution was 3.4 mm 3.4 mm on a 64 64 matrix, with 10 slices of 5 mm thickness . Other sequence parameters included tr / te / alpha = 2 s/19 ms/90 and ti1/ti2 = 700 ms/1400 ms . A slab thickness of 200 mm was used, with a 10 mm gap between the top of the label slab and the most inferior image slice . Mr signals were received using an eight - channel receive - only head coil, with excitation and labeling performed using the system body coil . These scans were at 1 mm isotropic resolution, acquired using an mprage sequence with ti / tr / te / alpha = 900/2300/2.94/9 . Voxel size was 1.0 1.0 1.2 mm . Flow and bold images were generated using the surround subtraction the sequence of flow images was generated by computing the difference between each image and the average of the previous and subsequent images . The sequence of bold images was computed by adding each image to the average of its two neighbors . For each run, the effect and standard error maps were then generated by fitting a linear signal model to each voxel in the flow and bold series . The model consisted of a term representing the three task epochs in the run convolved with a dual gamma function including positive response plus undershoot, plus a third - order polynomial . Multiple runs for each subject were then combined using a mixed - effects model, followed by spatial normalization to the mni 152 brain and combination of normalized maps for different subjects again using a mixed - effects model (as described in worsley et al . ). Individual and multisubject maps were then thresholded at p = .001 significance with correction for multiple comparisons using the stat_threshold routine of the fmristat software package . Regions of interest (rois) were generated using the neurolens software package (www.neurolens.org). Average bold statistical maps for each subject were used to make a v1 roi by thresholding as described above . Voxels exceeding threshold in the bold map but located outside the banks of the calcarine sulcus as visualized in the t1-weighted structural scan were manually edited from the roi, to ensure that the signals extracted were associated with primary visual cortex and therefore contained tissue organized as ocular dominance columns . The effect values were then averaged within the roi for each functional scan and tabulated as effect size standard error . Values were converted to percent change as needed by dividing the effect size by the constant (dc) term fit during linear modeling and multiplying by 100 . Figure 1 shows mixed - effects bold and flow maps over all subjects for monocular and binocular stimulation . Occlusion of input to one eye reduced the amount of activation detected in extrastriate areas . However, the significance levels observed within primary visual cortex appeared similar during both monocular and binocular stimulation . Averaged time course signals for flow and bold are shown in figure 2, revealing the initial bold signal overshoot and poststimulus undershoot commonly observed in visual cortex during checkerboard stimulation (seen here during both monocular and binocular stimulation). The flow signal illustrates the lower signal - to - noise ratio generally obtained in arterial spin - labeling measurements . The bar graphs in figure 3 show average percent changes in bold and flow signals within the v1 rois of all subjects . The average percent change in bold signal for monocular stimulation was 0.930.04, which was significantly (p <.05) less than the percent change of 1.11 0.05 observed during binocular stimulation . The percent flow increase measured during monocular stimulation was 29 2, also significantly less than the percent change of 37 2 observed during binocular stimulation . Expressed as a percent reduction in the response amplitude, withholding input from one of the two eyes resulted in a 16 6% decrease in the bold response and 19 9% decrease in the perfusion response . The percent changes in bold signal per percent signal increase in flow (i.e., the quotient%bold%flow) during monocular and binocular stimulation were found, respectively, to be 0.031 0.004 and 0.030 0.004 (figure 4). The difference between these ratios was not statistically significant, failing to support any difference in flow - metabolism coupling during the two forms of stimulation . In this study, we examined the coupling between bold and cbf responses in primary visual cortex during monocular and binocular stimulation . We found that the bold and cbf responses to monocular visual stimulation were both significantly reduced in v1 relative to the responses observed during binocular stimulation (figure 3). The ratio of bold to cbf effect sizes did not differ significantly between the two stimulation conditions (figure 4), indicating comparable coupling between flow and oxidative metabolism regardless of the columnar fraction that was activated . The results obtained in the present study do not support the strong form of the theory that tissue perfusion is regulated only on a coarse spatial scale, irrespective of the spatial precision with which metabolism might change . This notion has been described previously as watering the entire garden for the sake of one thirsty flower . To borrow the garden analogy, the experiments described here could be described as an attempt to measure the total water intake of the garden, as well as the runoff of unused water, to test this hypothesis . Our results are consistent with recent mri studies showing that there is in fact sufficient spatial contrast in the perfusion response, as imaged using arterial spin - labeling, to resolve columnar structures in the visual cortex . The study by duong et al . Found that the early negative bold response (initial dip) also exhibited a high degree of spatial localization, whereas the late positive bold response was more diffuse . It is important to remember that the apparent resolution of each signal is dictated both by the underlying physiological regulatory precision and by the degree to which confounding vascular structures are superimposed on the pattern of parenchymal activation . Based on our results and those from duong et al ., it appears likely that the lack of precision in the late bold signal is due primarily to obscuring effects from the macrovascular anatomy, rather than a diffuse parenchymal bold effect . It has been suggested by other authors that the increase in the apparent precision of the initial dip arises because bold signal increases in large draining veins do not arise until after the initial transit of blood through the local capillary bed postulated to take approximately one second . Given that functional signals of interest may exhibit bias due to vascular anatomy, designating regions of interest using objective criteria is an important part of quantitative neuroimaging studies . In the present study, the use of phase - encoded retinotopic mapping to identify v1 in a separate mapping experiment would have been the optimal approach, since this procedure yields a set of voxels exhibiting a specific spatial trend in the polar angle or eccentricity of their projection in visual field that is unlikely to appear in a large vein . This would have led to excessively long scan sessions, however . Instead, we used the fact that the optic radiations terminate in the calcarine sulcus, making it very probable that activated regions lying in this anatomical zone are in fact part of primary visual cortex . It is still possible that the bold activation maps used to create rois based on a simple activation minus baseline contrast could contain a disproportionate contribution from large draining veins . These veins mix venous outflow from multiple visual areas, including regions which do not exhibit eye - specific columnar segregation, diluting any shift in flow - bold coupling present specifically in v1 . In a pilot study of six subjects performed at 1.5 t using retinotopic mapping to identify v1 (but performed using single - slice asl at 1.5 t) we therefore do not feel that the results of the present study are substantially impacted by our roi selection procedure . Moreover, the relatively large voxel size and intense stimulus used in the present study yielded diffuse regions of robust activation that did not appear to be limited to macrovascular responses (as can occur at higher spatial resolutions or with weaker stimuli). By measuring total flow and bold responses in v1 during activation of different columnar fractions, we were able to achieve high signal - to - noise ratios (snrs) compared to studies that have used extremely high spatial resolution to actually resolve the columns . The purpose of the present study was to provide insight into two questions: the first is whether there is in fact a fundamental difference in the spatial precision with which perfusion and oxidative metabolism is measured; the second was to determine the impact of partial activation of a cortical columnar system on the bold signal characteristics observed at a customary fmri spatial resolution . If there is indeed a profound mismatch in the spatial extent of increases in oxidative metabolism and flow, this should be apparent in the total average signal over v1 . That none was found suggests that similar extents are likely to be found at higher spatial resolutions . However, it is notable that while removal of input from one of the two eyes did result in a reduction of both bold and flow signals, the response decreased by much less than one half . This is consistent with detailed autoradiographic studies showing that pronounced ocular dominance segregation is mainly limited to cortical layer iv, with layers ii and iii actually exhibiting higher activation during binocular than monocular stimulation [10, 18]. This is consistent with later human neuroimaging studies, in which some regions showed higher apparent activity levels during the appropriate monocular stimulation than during binocular input [16, 19]. The columnar structure associated with ocular dominance is therefore most appropriately viewed as reflecting a moderate bias in overall selectivity, associated primarily with a single cortical sublayer, superimposed on numerous other modulating influences . In light of the issues discussed above, it is clear that the columnar segregation of brain activation is not all or nothing during selective stimulation such as monocular or single - orientation conditions . Much of the early research in this area, performed using optical imaging methods capable of producing compelling maps of the columnar architecture, examined the perfusion of orientation domains (e.g., malonek and grinvald) and not ocular dominance columns although a number of authors have imaged ocular dominance using a variety of other methods [5, 6, 10, 19, 20]. It would therefore be informative to replicate the present study using different combinations of oriented stimuli . It is also possible that certain stimuli might achieve a higher degree of selectivity than the ones used in this and prior studies . Perhaps under such conditions a small difference in flow - bold coupling might become detectable . Future investigation of this topic might include the use of different stimuli designed to selectively activate pathways involved in stereopsis . Our results do not support the theory of spatially decoupled responses in blood flow and oxidative metabolism during activation of a subset of cortical ocular dominance columns . The limited impact of monocular blockade on flow and bold response amplitudes is also demonstrated, and should serve as a caution that ocular dominance contrast is likely to be faint in hemodynamic imaging methods.
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Metabolically healthy obese (mho) term refers to obese individuals who are relatively insulin sensitive and normotensive and have favourable glucose and lipid profile inspite of high levels of obesity [1, 2]. Evidence suggests that mho individuals as based on bmi criteria may account for as much as 2030% of obese population [3, 4]. Mho subset of individuals is relatively protected from obesity related cardio - metabolic disturbances that increase cardiovascular disease (cvd) risk in metabolically abnormal obese . However, data concerning the exact risk of cvd in mho as compared to healthy normal weight individuals is limited, and most data are confined to women at narrow age range . Carotid artery intima media thickness (cimt) is a non - invasive surrogate marker of subclinical atherosclerosis and an indicator of cvd risk [6, 7]. In addition, imt is associated with age, male gender, obesity, and traditional risk factors [811]. Beside traditional cvd risk factors, obesity is associated with changes in insulin like growth factor-1 (igf-1) [12, 13] which may be linked to atherosclerosis . Previous studies concerning the association between igf-1 and atherosclerosis have led to conflicting results; some authors found positive association between igf-1 and imt in men and in both men and women, while others found inverse association between igf-1 and carotid imt in healthy women . Another study in middle aged subjects showed that imt and igf-1 are positively associated in women and inversely in men . Greater subclinical cvd burden in metabolically benign obese individuals compared to metabolically healthy lean women was recently reported . Further studying of subclinical cvd profile in mho men and women and investigation of its correlates may help in proper management of mho individuals . The aim of the present study was to assess carotid intima media thickness (cimt) as a marker of subclinical atherosclerosis in metabolically healthy obese (mho) subjects as compared to healthy nonobese subjects . Also we evaluated the relation of cimt with age, sex, and igf-1 in mho subjects . The study comprised 75 mho subjects (37 men and 38 women, mean age 48.85 11.59 years) and 80 age, and sex, matched healthy nonobese (bmi 2024.9 kg / m) control subjects (table 1). Healthy obese adults were included in the study, and they were recruited from obesity clinic of specialized medical hospital, mansoura university, egypt . All subjects signed an informed consent to be included in our study . Exclusion criteria included smoking, alcohol consumption, cardiovascular, inflammatory or metabolic diseases, known or suspected pituitary, thyroid, adrenal or gonadal dysfunction, pregnancy, abnormal liver function tests, and medications (antihypertensive, lipid lowering, hypoglycaemic agents, growth hormone, anabolic steroids, glucocorticoids, hormone replacement, and hormonal contraception). Anthropometric measurements including height, weight, body mass index (bmi), and waist circumference (wc) were obtained using standardized techniques; height was measured to the nearest 0.5 cm, body weight was measured to the nearest 1.0 kg using mechanical weight scale (in the fasting state in the morning while wearing light clothes and no shoes). Bmi was calculated as weight / height (kg / m), and wc was measured at the highest point of the iliac crest at the end of normal expiration . Blood pressure (bp) was measured in the sitting position, with a mercury sphygmomanometer after a 10-minute rest . The systolic and diastolic blood pressure were read to the nearest 2 mmhg and recorded at the appearance (phase i) and disappearance (phase v) of korotkoff's sounds respectively . Metabolically healthy state, defined as having 0 to 1 out of six cardiometabolic abnormalities including (1) bp 130/85, (2) fasting triglyceride level 150 mg / dl, (3) hdl - c <40 mg / dl in men and <50 mg / dl in women, (4) fasting glucose 100 mg / dl (atp - iii cut - off values), (5) homa - ir> 2.7, (6) high sensitive c - reactive protein (hs - crp)> 3 mg / l (cutoff values for homa - ir and hs - crp were obtained from karelis and rabasa - lhoret). Fasting blood sample (10 ml) was drawn from cubital vein of each participant in the sitting position between 8.00 and 10.00 a.m., after 12 hours of fasting . Each blood sample was divided into two tubes, one of them containing edta for preparation of plasma and the other for preparation of serum . Plasma glucose and serum lipids were assayed immediately, and the remaining of the samples were kept in aliquots at 70c till the time of the rest of assays . Fasting plasma glucose (fpg) was estimated using commercially available kit supplied by human (germany). Fasting serum insulin was assayed by a solid - phase, enzyme - labeled chemiluminescent immunometric assay using immulite analyzer supplied by siemens (usa). Homeostasis model assessment of insulin resistance (homa - ir) index was calculated according to the formula: homa - ir = fasting insulin (u / ml) fasting glucose (mmol / l)/22.5 . Total cholesterol (tc), triglyceride (tg), and high density lipoprotein cholesterol (hdl - c) were assayed by commercially available kits, cobas (integra-400) supplied by roche diagnostic, germany . Low density lipoprotein cholesterol (ldl - c) was calculated according to friedewald et al . . High sensitivity crp (hs - crp) was estimated using an immunoenzymometric assay supplied by monobind inc . Serum igf-1 was estimated by immunoenzymometric assay supplied by roy bio (usa) with intraassay cv <10%, inter - assay <12%, and no cross - reactivity with other cytokines . Carotid imt was measured by b mode ultrasound using high frequency transducers 47 and 512 mhz; all subjects were examined with atl 5000, usa, and philips 11xe, the netherlands . Subjects lie supine with slight head tilt to the contralateral of the examined side, with elevation of the shoulder to stretch the neck in subjects with short neck . Common carotid artery (cca) was scanned bilaterally (in the longitudinal and transverse views) 1 cm before carotid bulb over a length of 1 cm at the far wall of both ccas . Imt thickness was measured at plaque free segments only as the distance between the leading edge of the lumen - intima interface and media - adventitia interface . Two measurements were taken from each side and averaged; then, the mean carotid imt was calculated as the average of measurements obtained from both ccas (figure 1). Student's t - test was conducted to compare the mean of continuous variables in two groups . Baseline characteristics of the mho subjects and nonobese healthy controls are shown in table 1 . Mho subjects had significantly higher sbp, fasting insulin, homa - ir, tg, ldl - c, crp, and cimt (0.78 0.15 versus 0.67 0.10) and significantly lower hdl - c and igf-1 (203.7 40.3 versus 229.4 57.5) than those of healthy nonobese subjects . Mho participants were divided into 2 groups according to sex . Compared to mho women, no significant difference was observed between mho men and women regarding fasting insulin, homa - ir, tg, hs - crp, and igf-1 (table 2). Cimt was significantly higher in mho men than in mho women (0.82 0.16 versus 0.74 0.14) (table 2). Mho women were divided into 2 age subgroups with respect to menopausal state: from 30 to 50 year (premenopausal) and above 50 years (after menopause), and they were compared to their age range matched subgroups of mho men; cimt was significantly higher in mho men age subgroup ranged from 30 to 50 years than that in their age ranging matched pre - menopausal mho women . However, the difference in cimt between mho men and women age subgroups above 50 years was insignificant (figure 2). The correlation between cimt and other parameters in mho subjects is shown in table 3 . Cimt was positively correlated with age, bmi, wc, sbp, homa - ir, tg, and ldl - c and negatively correlated with igf-1 . Regression analysis was done to identify predictors of cimt in mho (table 4). Age, sex, sbp, bmi, wc, homa - ir, tg, ldl - c, and igf-1 were entered in the regression model . Only middle age, male gender, wc, and igf-1 remained independently and significantly associated with cimt in mho subjects . The exact effect of metabolic healthy obesity on subclinical cardiovascular disease particularly in relation to age, sex, and different risk factors is not clearly defined . In the current study, we assessed cimt and its relation to age, sex and igf-1 in mho . The main finding was elevated cimt and reduced igf-1 in mho subjects; furthermore, imt in mho subjects was independently associated with male gender, middle age, and igf-1 . Our results are in agreement with previous findings of higher cimt in metabolically benign overweight / obese women compared to normal weight women . In addition rnlv et al . Confirmed that the risk of cvd and mortality is elevated in mho individuals compared to metabolically healthy and lean individuals . In contrast, park et al . Found similar cimt in healthy obese and healthy normal weight individuals . Despite relatively favourable cardiometabolic profile in mho as compared to metabolically abnormal one [1, 2], mho individuals appear to have greater subclinical cvd than normal healthy individuals . These data go in line with our findings of less favourable metabolic profile in mho as indicated by significantly higher fasting insulin, homa - ir, tg, and ldl - c, and significantly lower hdl - c . Obesity itself may contribute independently to carotid structure and function abnormalities and early atherosclerotic changes in obese are only partially explained by traditional cv risk factors . Obesity may modulate cca diameter and may induce adaptive changes in carotid wall thickness independent of metabolic and atherosclerotic factors . In addition to the increase in bp, obesity is associated with an elevation in total blood volume, basic cardiac output, and heart rate, which can induce new intima proliferation through changes in arterial wall stress . In addition, we found significant correlations between cimt and bmi, wc, sbp, tg, ldl - c, and homa - ir in mho subjects . However, among all previous cardio - metabolic variables, only wc was a significant predictor of cimt . Lakka el al . Demonstrated that abdominal obesity can be associated with progressive increase in cimt independently of general obesity and other risk factors . In healthy individuals, wc as a measure of abdominal obesity correlates better than bmi with subclinical atherosclerosis evaluated by cimt . De michele et al . Also found a significant association between whr and carotid wall thickening independent of fasting insulin concentration . We suggest that the presence of visceral obesity is more important than insulin resistance in healthy obese and the effect of insulin resistance may be confounded by wc, and some measured (igf-1) or unmeasured factors . Our findings suggest that abdominal obesity in mho individuals may exert an independent effect on atherosclerosis and it may mediate the effect of obesity on early atherosclerosis so, reduction of wc should be a target not only in metabolically abnormal obese subjects but also in mho subjects . Less favourable inflammatory profile indicated by significantly higher hs - crp levels was detected in mho subjects . We found insignificant relation between hs - crp and cimt . In agreement, sinning et al . Found no independent association between cimt and crp . It is suggested that crp is more predictive for cardiovascular prognosis in subjects of established carotid atherosclerosis . In addition, age was the most significant predictor of cimt among mho subjects; this is consistent with previous studies reporting an association between imt and age in both genders [8, 9]. Aging induces intrinsic changes in the arterial wall including progressive increase in intimal thickness, and this constant increase in imt over age is an effect present in both male and female . In accordance with a previously reported gender difference in imt, we found that mho men had significantly higher cimt than mho women, and the male gender remained independently associated with cimt; this can be explained in part by a small increase in ldl - c and decrease in hdl - c in our mho men as compared to mho women . Furthermore, the difference in cimt between mho men and women in the current study seems to be influenced by menopausal status because it was more significant between middle age, than older age, subgroups . Tan et al . Reported that traditional risk factors explain only a small amount of gender variance in imt, supporting the hypothesis that other sex hormone - related, behavioural, or genetic factors may play a role in the gender differences . Igf-1 is a nontraditional factor which has been linked to atherosclerosis and obesity [12, 34]. We observed that mho subjects had lower igf-1 levels than healthy nonobese subjects which goes in line with the negative association between igf-1 and bmi found by others [12, 13]. The relation between obesity and igf-1 is complex; it is hypothesized that free igf-1 increases with body weight until it reaches a level that triggers a negative feedback that would suppress growth hormone secretion (gh) which in turn would result in decreased igf-1 production in the liver . These results are parallel to those of marini et al ., who suggested that low igf-1 levels could contribute to early atherosclerosis in mho women compared with non - obese women . On the other hand, increased risk of atherosclerosis among individuals with low igf-1 was previously reported . In contrast, some found positive association between igf-1 and imt in men and in both men and women . Another study in middle aged subjects showed that imt and igf-1 are positively associated in women and inversely in men . This seems to be a conflict, however the relation between igf-1 system and cvd is complex, and there are many positive and negative associations . Both disorders of gh / igf-1 axis activity including gh deficiency characterized by low serum igf-1 and acromegaly accompanied by igf-1 excess are associated with increased intima thickness [36, 37], so, a probably u - shaped association between the gh / igf-1 axis activity and atherosclerosis was suggested though direct mechanisms of such a relation need to be elucidated . Some speculate that igf-1 is a proatherogenic by exerting an antiapoptotic effect on vascular smooth muscles and endothelial cells, and by being a promigratory factor, however, a detailed atheroprotective effect of igf-1 has been fully reviewed . Atherosclerosis is a chronic inflammatory disease initiated by oxidative stress, so the association of low igf-1 with early atherosclerosis in our study is consistent with its potential anti - inflammatory and antioxidant effects; as well as its possible role in endothelial function through its stimulation of endothelial nitric oxide production [41, 42]. Interestingly, physical activity is an important regulator of gh / igf system, also weight loss restored low levels of free igf-1 to normal, this further highlights the beneficial effect of life style based weight reduction in healthy obese . Our study has some limitations, first, the cross sectional design, second, the sample size being relatively small; however, further prospective evaluation on larger scale study may be recommended . Third, cimt was assessed by one investigator; however, a reasonable examination protocol was done by an expert radiologist . In conclusion, cimt is elevated and igf-1 is reduced in mho subjects, and cimt is independently associated with male gender, middle age, and igf-1.
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So many authors have studied clinostomum parasites, for example, the few recent studies like (14) are well documented . However, record on the occurrence of co - infection among three clinostomum species is unknown . The present study is the first record of co infection of three clinostomum species and report on the parasites of opi lake . 29 35 e is a tropical freshwater lake located in the valley of river uhere, northeast of nsukka, nigeria . The lake is about 300 meters from uhere river, the soil is porous and subject to severe erosion (5). However, the best knowledge about the lake was information on the systematic and basic biology of the species of organisms other than parasites found there . For instance, the climate, hydrobiology, macro invertebrates, and vegetation of the dominant flora have been described (69). Therefore, investigation into its parasites and description of co infection by three clinostomum species was necessary . Species of tilapia zilii (10) were collected using multiple fishing gear; cast nets, hook and line, and seine nets (150 mm 200 mm) monthly for twelve months from nov 2007 oct 2008 . Rank - abundance and species diversity of the parasites were determined using quantitative analysis of shannon - wienner index (14). Three metacercariae of clinostomatids were discovered to be the parasites associated with the t. zilii of opi lake . The parasites include: clinostomum tilapiae ukoli, 1966, c. complanatum rudolphi, 1819, and euclinostomum heterostomum rudolphi, 1809 . C. complanatum (9.4%), e. heterostomum (10.4%), and c. tilapiae (4.8%) with mean intensity; c. complanatum (4.2), c. tilapiae (4.6), and e. heterostomum (2.1) (table 13). Out of 392 t. zilii examined, 38 were infected with c. complanatum; c. tilapiae 19, and 41 hosts had e. heterostomum . C. complanatum and c. tilapiae were equally abundant (pi 0.26), while e. heterostomum was most abundant (pi 0.48) (fig . 1). Three major microhabitats buccal cavity, eye, and skin were seriously affected . In addition, the infection of the buccal cavity had the highest infection than the other two microhabitats of the eye and skin . Implications of their presence caused pronounced inflammation as well as roughening of the skin by the encysting metacercariae . Out of 85 e. heterostomum only 15 were not excysted, 57 c. tilapiae and 112 of c. complanatum were encysted respectively . These excysted forms caused serious damages to the infected fish as they burrowed through the organs of the host . Damages to the skin, blindness and bumps on the skin could affect the palatability and marketability of the infected fish as well as the acceptance of fish as the primary source of animal protein . For instance, nigeria with population of over 120 million individuals is the largest consumer of fish in africa and fish serve as primary source of animal protein to this huge population . The crowding population of opi around the lake is also prone to the inflammation of the naso - pharynx known as pharyngitis . This is because ingestion of under cooked fish with clinostomum infection results in the attachment of the trematode on the pharynx . Spatial adaptation of theses clinostomatids in the various microhabitats of t. zilii indicate selection for relatively better adaptiveness, and host location . Consequently, this resulted in a trade - off among excysted individuals, encysted individuals, damages to organs by scavenging large population size of the parasites in the host . Adaptation is a heritable trait that either spread because of natural selection or has been maintained by selection to the present or currently spreading relative to alternative traits because of natural selection . In all such cases, the trait in question has conferred and continues to confer or is just beginning to confer higher genetic or reproductive success on c. complanatum with highest population than the other two species of the parasites . Even with more encysted forms, adsorption of nutrients such as free proteins, amino acids and transaminases was readily very successful (15). Fitness is a measure of an individual s reproductive or genetic success, so that fitness benefit refers to the positive effect of a trait on the number of surviving offspring produced by an individual or the number of genes it contributes to the next generation whereas fitness cost refers to the damaging effects of the trait on these measures of individual genetic success (16). Although, encysted larvae can feed from the host, excysted larvae could be more voracious feeder . These suggestive pressures on natural selection would favor e. heterostomum in t. zilii during the cause of time and possible domination in the fish hosts in this freshwater lake . Also, because the parasites do not depend on stored food of the host; the nutrients they can obtain from their intermediate hosts are sustainable . It might last throughout the lives of the host fish due to difficulty in locating their definitive hosts, piscivorous birds such as egreta egreta (17). Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.
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Previously, we identified a brain specific nuclear gtpase called pike, which binds pi 3-kinase and enhances its kinase activity . Ngf treatment elicits the membrane - associated 4.1n nuclear translocation and binds pike, which was initially identified as a 4.1n binding partner in a yeast two - hybrid screening . The binding of 4.1n to pike prevents its interactions with nuclear pi 3-kinase, leading to diminish the activation of pi 3-kinase by ngf 1 . Pike gtpase is activated by nuclear translocated phospholipase c-1 (plc-1), which acts as a guanine nucleotide exchange factor (gef) for pike . This action is not dependent on plc-1's lipase activity but appears instead to involve its sh3 domain 2 . Recently, we have identified and cloned a few isoforms of pike: pike - l and pike - a . Both of them contain an arfgap domain and two ankyrin repeats domains . The c - terminal protein structure resembles that of centaurin family, which are gtpase activating proteins (gaps) for arfs or have homology to arfgaps and arf effectors 3, 4 . In rat brain and hippocampal neurons, pike - l physiologically binds to homer, an adaptor protein known to link metabotropic glutamate receptors (mglur i) to multiple intracellular targets including the inositol 1,4,5 trisphosphate (ip3) receptor . The activation of mglur i by its agonists enhances formation of an mglur i - homer - pike - l complex leading to activation of pi 3-kinase activity and prevention of neuronal apoptosis 5 . Pike - a differs from pike - l and -s by lacking the n - terminal proline - rich domain (prd), which binds pi 3-kinase . Interestingly, pike - a directly binds to activated akt but not pi 3-kinase in a guanine nucleotide - dependent way and stimulates the kinase activity of akt . Pike - a is co - amplified with cdk4 in a variety of human cancers . The overexpressed pike - a enhances akt activity, promotes cancer cell invasion and prevents apoptosis 6 . Mounting evidence demonstrates that akt is a crucial regulator of diverse cellular processes and contributes to cancer progression . Activation of akt is essentially dependent on pi 3-kinase signaling . Nevertheless, pike - a - mediated akt activation is independent of pi 3-kinase . Therefore, pike - a provides a new mechanism whereby growth factors and other cellular stimuli regulate akt, and functions as an oncogenic effector of cell invasion . Here, we discuss the recent advances in biochemical and molecular studies and future directions that might further our understanding of the roles of nuclear pi 3-kinase and pike family . Pike - s comprises 753 residues, containing three proline - rich domains (prd) in the n - terminus, followed by a gtpase domain and a partial pleckstrin homology (ph) domain in the c - terminus . Ngf treatment of pc12 cells activates pike gtpase with peak at 30 min and decays after 1 h. pike - s binds nuclear pi 3-kinase in a gtp - dependent manner . Pike - s activates pi 3-kinase depending on the presence of its two subunits with pike - s being able to bind independently to each of the two subunits p85 and p110 . Transfected by itself, p110 lacks catalytic activity but acquires it following co - transfection with p85 . Pike - s can activate pi 3-kinase only when both p110 and p85 are expressed, not with p110 alone 1 . Normally, gtpases cycles between the active, gtp - bound and inactive, gdp - bound states 7, 8 . Guanine - nucleotide - exchange factors (gefs) act as positive regulators that promote release of gdp and consequent formation of the active gtp - bound state, whereas gtpase - activating proteins (gaps) act as negative regulators, stimulating the intrinsic gtpase activity to generate the inactive gdp - bound form . Indeed, plc-1 binding to pike involves the sh3 domain of plc-1 interacting with the prd of pike - s 2 . We have demonstrated that plc-1 acts as a physiologic guanine nucleotide exchange factor (gef) for pike - s . This gef activity is mediated by the sh3 domain with phospholipase catalytic activity not required . Moreover, the sh3 domain but not phospholipase catalytic activity of plc-1 is necessary for its mitogenic actions, probably through activating the nuclear pi 3-kinase 2 . The activated plc-1 translocates to the plasma membrane and triggers the hydrolysis of phosphatidylinositol 4,5-bisphosphate (pip2) into two second messengers, inositol 1,4,5-triphosphate (ip3) and diacyglycerol (dag), which regulate the intracellular caand activation of protein kinase c respectively . Plc-1 does not possess any known nuclear localization signal and predominantly localizes in the cytoplasm . Nevertheless, it has been reported to be present in the nucleus 9 - 12 . It has been shown that plc-1 localizes to the nucleus of highly transformed and proliferating cell lines (a431, hela, mouse hepatoma mh 22a, rat zajdela ascitic hepatoma) but not to the nucleus of primary embryo skin or lung fibroblasts, where the enzyme is primarily cytoplasmic . Differential subcellular localizations in normal or highly transformed cell lines may reflect the degree of transformation of the cell type or phase of the cell cycle 10 . This finding is consistent with the observation of an increased amount of plc-1 in nuclei of 22 h regenerating rat liver, suggesting a relationship between the s - phase of the cell cycle and intranuclear localization of plc-1 13 . Pi 3-kinase is a key regulator of many cellular processes including cell proliferation, survival, motility, vesicular trafficking and carbohydrate metabolism . Pi 3-kinase is activated by both receptor tyrosine kinases (rtk) and cytoplasma membrane associated gtpase proteins . The activated pi 3-kinase generates the critical second messengers: the d-3 position phosphorylated phosphoinositides, which bind to the pleckstrin homology domain (ph) of numerous signaling molecules including phosphoinositol - dependent kinase 1 (pdk1), akt / pkb and plc-1, etc . While much has been learned about pi 3-kinase signal transduction in the cytoplasm, almost nothing is known about it in the nucleus . Previous studies show that pi 3-kinase also occurs in the nucleus 1, 14 - 19 . Stimulation of cells with ngf activates nuclear pi 3-kinase with nuclear accumulation of 3-phosphorylated phosphoinositide lipids 18, 20 . However, the physiological role of nuclear pi 3-kinase and its mediated signaling pathways remain obscure . The interaction between pike - s and pi 3-kinase is gtp - dependent like that between ras and pi 3-kinase 1, 21 . Ngf treatment of pc12 cells activates pike and nuclear pi 3-kinase with peaks at 30 min, whereas ngf activates the cytoplasmic gtpases of the ras family as well as cytoplasmic pi 3-kinase much more rapidly with peak activity in 5 - 10 min 21 . Moreover, in dominant - negative pike - s (k413as414n) retrovirus infected pc12 cells, activation by ngf of nuclear pi 3-kinase is abolished, suggesting that pike - s is the major mediator of nuclear pi 3-kinase . Cytoplasmic pi 3-kinase activation requires activated receptor tyrosine kinases (e.g. Pdgfr, egfr, cd28, etc .) Or gtpase proteins such as ras . However, none of these known pi 3-k activators are present in nucleus . Our discovery that the nuclear gtpase, pike, enhances nuclear pi 3-kinase activity indicates that pike - s may be the nuclear counterpart of ras . The extreme n - terminus of pike - s associates with the c - terminal domain (ctd) of protein 4.1n, a neuronal isoform of the erythrocyte membrane cytoskeletal protein 4.1r . Therefore, activation of nuclear pi 3-kinase by pike is inhibited by the ngf - stimulated 4.1n translocation to the nucleus 1 . Plc-1 and pi 3-kinase share the same substrate pi (4,5) p2, and both enzymes are concomitantly recruited to the plasma membrane and activated, where they mediate each other's enzymatic activity . Several studies have suggested cross - talk between plc-1 and pi 3-kinase in the cytoplasm . For example, pi (3,4,5) p3 generated by pi 3-kinase influences plc-1 membrane translocation and activation by binding to its ph domain and a c - terminal sh2 domain 22, 23, and activation of plc downregulates pi 3-kinase by at least two mechanisms: (1) inhibition of irs-1-associated pi 3-kinase; and (2) acute activation of a ptdins (3,4,5) p3 5-phosphatase . Ngf elicits plc-1 nuclear translocation and acts as a gef for pike through it sh3 domain . Thus, the nuclear plc-1/pike / pi 3-kinase signaling pathway seems to be the extension of the cross - talk between the cytoplasmic plc-1 and pi 3-kinase . Pike - l was identified in searching databases for sequences that might resemble pike - s . Pike - l differs from pike - s in containing a 40 kda c - terminal extension which includes an arf - gap and two ankyrin repeats domains 5 . Pike - l and pike - s are alternatively spliced isoforms and brain - specific . However, whereas pike - s occurs in all brain regions examined, pike - l is uniquely absent from the cerebellum . Pike - s is exclusively nuclear, whereas pike - l occurs in multiple subcellular fractions and, by immunohistochemistry, is observed throughout the cell body and all neuronal processes 5 . Sequence analysis led to the discovery that pike - l binds to homer 1c, an adaptor protein localized to postsynaptic densities coupling cytoplasmic regions of group i coupling metabotropic glutamate receptors (mglurs) to inositol-1,4,5-trisphosphate receptors (ip3rs) as well as shank proteins 24 . The mglurs comprise three groups: group i (mglur 1 and 5), group ii (mglur 2 and 3) and group iii (mglur 4, 6, 7 and 8). Via g proteins, group i receptors stimulate phospholipase (plc) leading to the formation of ip3 and calcium mobilization . By contrast, group ii and group iii receptors are negatively coupled to adenylyl cyclase 25 . One particularly prominent action of group i mglurs is to protect neurons from apoptotic death 26 . Binding of mgluri with homer and thereby with pike - l occurs only with the group i class of mglurs, which are the only forms that contain the homer ligand pxxf motif . Amino acids 187 - 190 of pike - l are pkpf which corresponds to a domain recently appreciated as a consensus motif (pxxf) present in proteins that bind to the evh1 domain of homer 24 . Pike - l and homer 1c coprecipitate robustly, and the interaction is dependent upon the portion of pike - l containing the pkpf sequence . Mutation of proline-187 of pike - l abolishes binding of pike - l to homer 1c and provides a useful tool to analyze the importance of this binding in various signal cascades . Pike - l / homer 1c complex couples pi 3-kinase to metabotropic glutamate receptor 1 (mglur1), suggesting that mglur1 might activate pi 3-kinase separately from its well - known activation of phospholipase c. this pathway has been verified by the demonstration that mglur5 transfection of hek293 cells stimulates pi 3-kinase activity while mutants of mglur5 that do not bind homer fail to activate pi 3-kinase 5 . Moreover, homer mutants that do not bind mglur5 block pi 3-kinase activation . In hippocampal cultures mglur i activation increases pi 3-kinase activity, while infection with an adenovirus containing a dominant - negative form of pike - l blocks such activation . Delivery into hippocampal cultures of the homer - binding motif of pike as a dominant - negative, blocks mglur i stimulation of pi 3-kinase, while mutant peptides that do not bind homer have no effect . Finally, mglur i activation fails to stimulate pi 3-kinase in the cerebellum, a brain region that is devoid of pike - l . Prevention of neuronal apoptosis is a major action of group i mglurs, and this process appears to be mediated by pike - l and pi 3-kinase 5 . In hippocampal cultures mglur i activation and pike - l transfection block the apoptotic effects of staurosporine and other agents . By contrast, infection with a pike - l dominant - negative construct augments apoptosis, and a pike construct which cannot bind homer prevents the anti - apoptotic actions of mglur activation . Additionally, mglur i activation does not block apoptosis in the cerebellum, which lacks pike - l . Pike - l may have other activities besides mediating actions of mglur i on pi 3-kinase and apoptosis . Some possibilities are suggested by the structure of pike - l, whose c - terminal region contains an arf - gap domain and two ankyrin repeats . Gtpases are typically activated by gap proteins, but whether the arf - gap domain functions as an internal gap for the gtpase activity of pike - l remains to be determined, though there are instances of arf - gap domains exerting such activity 27 . Mediation of anti - apoptotic actions is the only mglur i function thus far examined in connection with pike - l . Group i mglurs exert a variety of neuronal functions, influencing motor learning and coordination via cerebellar purkinje cells 28 and influencing long term potentiation in the hippocampus 29 . In our evaluation of pike amplification in human glioblastoma multiforme, we have identified a third pike form (pike - a), which was originally identified in the human genome sequencing effort as kiaa0167 30, and was independently identified by liu and collaborators 27 . The chromosome 12q13 - 15 region is frequently amplified in human sarcomas and brain tumors 31, 32 . 12q13 - 15 amplicon contains two separate core regions, one containing mdm2 and the other containing cyclin - dependent - kinase 4 (cdk4) 32, 33 . Gene structure analysis reveals that pike - a results from different transcription initiation site from that of pike - l and -s . Pike - a contains the gtpase, ph, arfgap and two ankyrin repeats domains present in pike - l but lacks the prd containing n - terminus, which binds protein 4.1n, pi 3-kinase and plc-1 . Fluorescent in - situ hybridization (fish) and northern blotting analysis reveal that pike gene is amplified in a variety of human sarcoma and brain tumor cell lines 6 . Unlike pike - l or -s, pike - a does not associate with pi 3-kinase, instead, it binds to active but not inactive akt . Truncation analysis demonstrates that gtpase and ankyrin repeats domains interact with partial catalytic and regulatory domains of akt . Binding by akt to purified recombinant gst - pike - a is evident in cells treated with the mitogenic stimulus igf-1 compared to untreated cells . Gtp--s and gdp--s strongly stimulate binding of activated akt and pike - a compared to faint binding in the absence of guanine nucleotide treatment 6 . Interestingly, dominant - negative pike - a - dn binds akt promiscuously even in cells not treated by growth factor igf-1 and, in treated cells, binding occurs even in the absence of guanine nucleotides . Pike - a strongly binds to active akt, indicating that akt phosphorylation might play a role in mediating its association with pike - a . In vitro akt kinase assay demonstrates that pike - a substantially enhances akt activity, for which gtpase domain but not ankyrin repeats domain is responsible . To date, numerous pike binding proteins have been identified and are now depicted in figure 3 . Functional analysis of the interaction between pike - a and akt demonstrates that pike - a mediates invasion of cancer cells through akt . Two human glioblastoma cell lines, ln - z308, in which pike - a is heavily overexpressed, and u87 mg that does not overexpress pike - a, were utilized to dissect the physiological consequence of pike amplification . Infection of u87 mg cells with an adenovirus containing wild - type pike - a elicits almost a doubling of invasive cell number . Only a modest increase occurs with the ln - z308 cells, presumably because endogenous pike - a is already highly expressed . In both ln - z308 and u87 mg cells, infection with pike - a - dn reduces cell invasion by about 50% compared with control cells . Thus, pike - a is a physiologic regulator of akt and an oncogenic effector of cell invasion 6 . More recently, we show that overexpression of pike - a wild - type but not dominant - negative mutant prevents apoptosis through mediating akt activity . Collectively, these observations suggest that pike - a amplification contributes to cancer cell survival and progression by inhibiting apoptosis through upregulating akt 34 . Agap1/ggap1/kiaa1099 (accession number ab029022), m - rip (accession number af359283), and agap2/ggap2/kiaa0167, we have now designated it as pike - a (accession number d79989), consist of the identical gene structure . Ggap1 and ggap2 share approximately 50% sequence homology, while ggap1 and mrip1 share 70% sequence homology at the amino acid level . The complete protein sequences of this family encode an n - terminal ras - related gtpase domain, a ph domain, followed by a c - terminal gap domain and an ank repeat domain . The gtpase domain in the ggap proteins reveals 60% homology to the ras family of gtpases . Identical to k - ras, agap2 is 42% similar and 28% identical to rab9, and m - rip is 47% similar and 27% identical to h - ras 35 . Northern blotting analysis demonstrates that ggap1 mrna was found in most of the tissues although the level of expression is varied, with enriched expression in skeletal muscle, brain, placenta, and kidney . Most tissues contain two messages at 5 kb and 8.5 kb, while the peripheral blood leukocytes (pbl) have only one message with the size of 5.5 kb . The expression of pike - a is highly enriched in the brain, heart, skeletal muscle, and immune tissues 27, 32 . Northern blotting analysis of human brain regions reveals that a prominent band was found in cerebellum, cerebral cortex, occipital pole, frontal lobe, temporal lobe, and putamen for ggap1 and ggap2 (pike - a), while low level of expression was detected in medulla and spinal cord for pike - a 27 . Subcellular localization reveals agap / ggaps are expressed in different cellular compartments . In transfected cos-7 cells, flag - tagged agap1/ggap1 staining occurs in the cytosol, possibly in the internal membrane systems, such as the endoplasmic reticulum (er) and the golgi apparatus . On the other hand, the flag - tagged ggap2/pike - a was stained in both the cytoplasm and the nucleus this observation is consistent with the subcellular distribution of amplified pike - a in human glioblastoma cells 6 . Furthermore, cells overexpressing agap1/ggap1 are more flat and have much more lamellipodia than control cells . In contrast, cells overexpressing agap1ggap2/pike - a are smaller and more rounded than control cells . Although the similarity of the gtpase domain in agap1/ggap1 to k - ras, the purified recombinant protein does not bind to gdp, gtp, adp, or atp . No gtpase activity was detected in full - length agap1 or the isolated gtpase domain . Overexpressed agap1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and rab4 35 . Thus, agap1 is a phosphoinositide - dependent arfgap that impacts both the endocytic compartment and actin, consistent with the observations described 27 . Different from the findings on agap1/ggap1 by nie et al, xia and his colleagues demonstrated gtpase domain of agap1/ggap1 and ggap2/pike - a binds to [p]gtp, but the c - terminal segment of the proteins fail to display gtp - binding . Moreover, they showed the prominent gtpase activity from the full - length agap1/ggap1 and agap2/ggap2/pike - a immunopurified from the cells, transiently transfected with the cdnas encoding the flag - tagged proteins into cos-7 cells . The turnover number of agap1/ggap1 and agap2/ggap2/pike - a for gtpase activity is similar to gtpase ras (0.01 min) 27 . Furthermore, the authors showed that the c - terminal arfgap domain could regulate the activity of the n - terminal gtpase domain via direct intramolecular interaction . Bacterially purified gtpase domains from agap1/ggap1 and agap2/ggap2/pike - a have very low intrinsic gtpase activity . Addition of the arfgap domain from the same protein (ggap1 or ggap2) significantly increased the gtpase activity 27 . Phosphoinositide lipids bind to ph domain and activate dynamin gtpase activity, which is important in dynamin function during vesicle budding 36 . To test this possibility that phosphatidylinositol lipids also affect pike gtpase activity, we treated pike - s with different phospholipids including phosphatidylinositol 4,5-bisphosphate (pi-4,5-p2) and phosphatidylinositol 3,4,5-trisphosphate (pi-3,4,5-p3), but failed to detect its activation, presumably because it just contains a partial ph domain . However, pike - l contains a full module of ph domain and displays high homology with phosphatidylinositol bi- or tri- phosphates . In fact, our recent data reveal that it selectively binds to pi (3,4)p2, pi (3,4,5)p3 and pi (3,5)p2 and this interaction mediates pike - l's gtpase activity (unpublished data). This observation raises an intriguing and provocative possibility that pike - l might regulates its own gtpase activity through a negative feed - back mechanism using the pi 3-kinase lipid product . Neuronal activity - regulated synaptic -amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (ampar) trafficking plays a critical role in synaptic plasticity, a molecular model underlying the learning and memory . Synaptic ampar insertion and removal from postsynaptic membrane mediates n - methyl - d - aspartate receptors (nmdar) and mglur i - dependent long - term potentiation (ltp) and long - term depression (ltd) 37, 38 . While numerous proteins coupling nmdar to ampar have been demonstrated, little is known the molecular partners that mediate the crosstalk between mglur i and ampar . We have previously shown that protein 4.1n, which binds to the cytoplasmic tail of ampar 39, interacts with the extreme n - terminus of pike 1 . Moreover, mglur i activation stimulates the formation of homer / pike - l complex, which couples pi 3-kinase to mglur i 5 . These observations support an intriguing notion that pike - l - homer interaction might couple ampa receptor to mglurs through pike - l association with the postsynaptic proteins 4.1n . Protein 4.1 are a family of multifunctional cytoskeletal components (4.1r, 4.1 g, 4.1n and 4.1b) derived from four related genes, each of which is expressed in the nervous system 40 . Mouse 4.1r knockout supports the notion that protein 4.1 plays an important role in the nervous system, since multiple disturbances in behavior indicate the cerebellar and hippocampal defects, correlating well with the absence of 4.1r in cerebellar granule cells, and hippocampal dentate gyrus cells 41, 42 . Previous studies indicate that proteins 4.1 exist in both presynaptic and postsynaptic density 39, 43, 44, 45, 46 . Immunohistochemical studies using a polyclonal 4.1n antibody revealed several patterns of neuronal staining, with localizations in the neuronal cell body, dendrites, and axons . In primary hippocampal cultures, mouse 4.1n is enriched at the discrete sites of synaptic contact, colocalizing with the postsynaptic density protein of 95 kda (a postsynaptic marker) and glutamate receptor type 1 (an excitatory postsynaptic marker) 43 . Previously, 4.1n has been shown to associate with the membrane proximal region of glur1 in vivo and colocalize with ampa receptors at excitatory synapses, where it might implicate in ampa receptors clustering and connecting to the actin cytoskeleton 39 . Conceivably, protein 4.1n may function to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin - actin - based cytoskeleton, integral membrane channels and receptors, and postsynaptic protein like pike - l . This finding conforms to our current model in postsynaptic glutamate receptors clustering and synaptogenesis mediated by homer - shank interaction by docking mglurs to the nmdars . Pike - s gtpase is activated by plc-1, which acts as a physiologic guanine nucleotide exchange factor (gef) for pike . The identification of pike - l and -a isoforms, which contain an arfgap domain, might shed light on the molecular mechanism how pike gtpase is negatively regulated . The arfgap domain possesses potent gap activity on arf gtpase and its internal gtpase domain . Moreover, this gap activity is mediated by phosphoinositol lipids through ph domain of pike, suggesting a negative feed - back mechanism between pike / pi 3-kinase singaling . Besides its anti - apoptotic role in mglur i cascade, pike - l might also implicate in synaptic plasticity by coupling mglur i to amap receptor . Pike - a may have even broader relevance resulting from its subcellular localization (in both the nucleus and the cytoplasm) and tissue distribution . Pike - a might contribute to cancer progression by promoting cancer cell invasion and survival through up - regulating akt . Diagram of three pike isoforms.pike-l, an alternatively spliced form of pike, which is several hundred amino acids longer than the original form of pike, designated as pike - s (bankit475414 (ay128689, human)). In addition to the gtpase and ph domains shared by pike - s and pike - l, pike - l contains an arfgap domain and two ankyrin repeats . Pike - a contains the gtpase, ph, arfgap and ankyrin repeats domains present in pike - l but lacks the proline - rich domains (prd) containing n - terminus, which protein 4.1n, pi 3-kinase and plc-1 bind to . Plc-1/pike - s / nuclear pi 3-kinase signalling: ngf treatment of pc12 cells provokes plc-1 nuclear translocation, and stimulates pike - s gtpase to bind gtp . Ngf also causes 4.1n to translocate to the nucleus over a period of hours, lagging behind the translocation of pi 3-kinase and the peak activation of pike elicited by ngf . The decline of activated nuclear pi 3-kinase, which coincides with the appearance of nuclear 4.1n, might involve 4.1n sequestering pike away from nuclear pi 3-kinase . The decline of pike's ngf - induced gtpase activation takes place at about the same time and so also may participate in the decline of nuclear pi 3-kinase . Pike binding proteins: protein 4.1n and pi 3-kinase share the same binding motif on the n - terminus of pike . Mglur i adaptor protein homer 1 interacts with pike through homer binding motif pxpf (a.a . Akt associates with pike - a through its gtpase and c - terminal ankyrin repeats domains.
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Odontogenic keratocysts (okcs) are the most common form of cystic lesions affecting the maxillofacial region . They are clinically aggressive lesions which are thought to arise from the dental lamina or its remnants . Okc is known for its rapid growth, and its tendency to invade the adjacent tissues including bone . It has a high recurrence rate, and is associated many a times with nevoid basal cell carcinoma syndrome (nbccs). Usually, multiple okcs occur as a component of nbccs with concomitant cutaneous, skeletal, ophthalmic, and neurologic abnormalities . Gorlin and goltz first described the spectrum of features associated with this syndrome in 1960; hence, it is also called gorlin - goltz syndrome . Multiple okcs have been known to occur in non - syndromic cases, though it is very rare . These multiple lesions may be the first manifestation of the nbccs or otherwise it may be because of the multifocal nature of okcs . We discuss the possibility that the current case is a partial expression of nbccs and briefly review the current trends in treatment of recurrent okcs associated with this syndrome . A 23-year - old male normally fit and well reported to the outpatient department of our hospital with a chief complaint of asymptomatic swelling and pus discharge from the left maxillary posterior region since 10 days . Intra - oral examination revealed a partially edentulous state; teeth 13, 14, 18, 27, 28, 37, and 47 were missing with slight expansion of the buccal cortical plate in the left maxillary posterior region . On exertion of pressure, a white creamy exudate expressed out of the area between 23 and 24, but there was no tenderness or bleeding on further palpation . Lesion in the left maxillary quadrant was associated with multiple impacted teeth and retained deciduous teeth . In the lower jaw, well - defined radiolucencies were associated with missing 37 and 47 which mimics dentigerous cysts . Diagnostic biopsy from the maxillary the patient was admitted to hospital, and enucleation of the cystic lesions was performed under general anesthesia and the surgical bed was treated with carnoy's solution . It was interesting to note that there were two separate entities in the right maxillary region with one of the lesion associated with the impacted canine and they were sent as two separate specimens . Four lesions (excluding the small lesion distal to tooth 36) were enucleated and sent for histopathological examination . The patient recovered uneventfully and was discharged the next day and is presently being kept under observation with regular follow ups at the interval of 3 months are being conducted . The histopathologic report revealed that three out of four lesions were okc and the other lesion which was associated with the impacted canine in the maxillary right quadrant was dentigerous cyst . Cystic lining of all three keratocystic lesions was parakeratinized stratified squamous epithelium of uniform 6 - 8-cell thickness . The lining epithelium consisted of well - defined columnar basal cells in a palisade arrangement and with polarized nuclei . The height of the epithelial cells and the number of nuclei they contained were reduced . Multiple okcs usually occur as a component of nbccs or gorlin - goltz syndrome, orofacial digital syndrome, noonan syndrome, ehler danlos syndrome, simpson - golabi - behmel syndrome, or other syndromes . Our patient was normally fit and well with no significant family history and had no features suggestive of these syndromes, such as basal cell carcinoma, skeletal or orofacial defects, stunted growth, bleeding diathesis, hyper extensible skin and hyper mobile joints, or other congenital anomalies associated with overall growth . In a study by brannon, 5.1% of 312 cases were associated with nbccs and 5.8% were accompanied by multiple keratocysts, but without any other features of the syndrome . It is suggested that a two hit mechanism may underlie the variable expression of nbccs and sporadic okcs . In nbccs, the development of basal cell carcinoma and keratocysts in the absence of nbccs reflects two somatic hits in which there are mutations of ptch within locally susceptible cells that ultimately result in allelic loss . The absence of all the manifestations of nbccs may be due to variability of the ptch gene expression . The biological behavior of okcs associated with nbccs is more aggressive and these cysts have higher recurrence rates (82%) compared with solitary keratocysts (61%). The higher recurrence rates are attributed to epithelial remnants of the cystic lining or satellite cysts left behind following surgery . A recurring okc can be a new cyst that originates from epithelial residue or a microcyst left behind in the overlying mucosa . This is reinforced by the fact that okcs can occur in bone grafts if the overlying mucosa is not excised . The term multiple cysts does not necessarily mean that the patient must have more than one cyst at a given time; rather it refers to occurrence of cysts over the lifetime of the patient . One of the interesting findings in our patient was the concomitant occurrence of dentigerous cyst associated with impacted canine along with okc in the maxillary right quadrant which is very rare finding in itself . It is a well - known fact that dentigerous cysts associated with unerupted teeth occur only in few individuals, whereas unerupted teeth are common occurrence . Histologically, parakeratinization, intramural epithelial remnants, and satellite cysts are more frequent among okcs associated with nbccs than in solitary keratocysts . In our patient, the lining of the okcs revealed the presence of parakeratinization with underlying connective tissue infiltrated by mild inflammatory cells and at some place with foreign body type of giant cell reaction indicating nbccs association [figure 2]. The cystic space of the maxillary right quadrant associated with the impacted canine showed non - keratinized stratified squamous epithelium with 4 - 6 cells thick which appears to be collision of okc and dentigerous cyst [figure 3]. Lining of odontogenic keratocyst collision of odontogenic keratocyst with dentigerous cyst in the management, the recurrent okcs are conventionally treated with enucleation or marsupilization with adjuvant chemical cautery using carnoy's solution and cryosurgery . Cryosurgery seemed to be very promising as documented by schmidt and pogrel where they have treated these lesions with enucleation along with liquid nitrogen cryotherapy . Electro - cauterization may also be used in these areas to eliminate possible remnants of the cyst wall that tends to tear in those areas . We have treated the lesions in our patient with enucleation followed by electrocautery and also with carnoy's solution . At present the patient is kept under observation with a regular follow up at an interval of 3 months . The patient and his relatives have been explained about the possibility of developing other features of nbccs in the coming years and are advised to keep a strict vigil of the same . In conclusion, any patient reporting with the multiple okcs should be evaluated thoroughly for the possibility of nbccs as okcs may be the first and only manifestation of this syndrome . Also for the fact that okcs associated with this syndrome have higher rate of recurrence than the isolated okcs, a very strict follow up has to be followed for a long period of time . The possibility of other features of nbccs has to be explained to the patient as well as his relatives, so as to allow appropriate genetic counseling and serial screening for the development of malignancies and other complications besides okcs.
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Fear of hypoglycaemia and gain in body weight act as barriers for initiation of insulin therapy . Modern insulin analogues are a convenient new approach or tool to glycaemic control, associated with low number of hypoglycaemia and favourable weight change . A1chieve, a multinational, 24-week, non - interventional study, assessed the safety and effectiveness of insulin analogues in people with t2 dm (n = 66,726) in routine clinical care . Please refer to editorial titled: the a1chieve study: mapping the ibn battuta trail . The patient characteristics for the entire cohort divided as insulin - nave and insulin users is shown in the table 1 . The majority of patients (77.8%) started on or were switched to biphasic insulin aspart . Other groups were insulin detemir (n = 1001), insulin aspart (n = 734), basal insulin plus insulin aspart (n = 117) and other insulin combinations (n = 189). Overall demographic data after 24 weeks of treatment, overall hypoglycaemic events reduced from 0.8 events / patient - year to 0.1 events / patient - year in insulin naive group and from 2.6 events / patient - year to 0.7 events / patient - year in insulin user group . The hypoglycaemia incidence in insulin naive group at 24 weeks was lower than that observed in insulin users at baseline . Blood pressure decreased whereas overall lipid profile and quality of life improved at week 24 in the cohort [tables 2 and 3]. All parameters of glycaemic control improved from baseline to study end in the total cohort [table 4]. Overall efficacy data of the total cohort, 7217 patients started on biphasic insulin aspart ogld, of which 5995 (83.1%) were insulin nave and 1222 (16.9%) were insulin users . After 24 weeks of starting or switching to biphasic insulin aspart, hypoglycaemic events reduced from 0.2 events / patient - year to 0.0 events / patient - year in insulin nave group and from 2.2 events / patient - year to 0.1 events / patient - year in insulin users group . Body weight decreased and quality of life improved after 24 weeks of treatment [tables 5 and 6]. Glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [table 7]. Biphasic insulin aspartoral glucose - lowering drug efficacy data of the total cohort, 117 patients started on basal + insulin aspart ogld, of which 58 (49.6%) were insulin nave and 59 (50.4%) were insulin users . After 24 weeks of starting or switching to biphasic insulin aspart, hypoglycaemic events reduced from 2.7 events / patient - year to 0.6 events / patient - year in insulin nave group and from 4.9 events / patient - year to 1.4 events / patient - year in insulin users group . Body weight decreased and quality of life improved after 24 weeks [tables 8 and 9]. Basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to basal + insulin aspart oglds for both insulin nave and insulin user groups [table 10]. Basal+insulin aspartoral glucose - lowering drug efficacy data of the total cohort, 1001 patients started on insulin detemir ogld, of which 865 (86.4%) were insulin nave and 136 (13.6%) were insulin users . After 24 weeks of starting or switching to biphasic insulin aspart, hypoglycaemic events reduced from 1.0 events / patient - year to 0.2 events / patient - year in insulin nave group and from 2.3 events / patient - year to 0.7 events / patient - year in insulin users group . A decrease in body weight and improvement in quality of life was observed at the end of the study [tables 11 and 12]. Insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir oglds for both insulin - nave and insulin user groups [table 13]. Insulin detemiroral glucose - lowering drug efficacy data of the total cohort, 734 patients started on insulin aspart ogld, of which 583 (79.4%) were insulin nave and 151 (20.6%) were insulin users . After 24 weeks of starting or switching to insulin aspart, overall hypoglycaemia decreased from 0.6 events / patient - year to 0.0 events / patient - year in insulin naive group and from 2.7 events / patient - year to 0.5 events / patient - year in insulin users group . A decrease in body weight and improvement in quality of life insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin aspart oglds for both insulin nave and insulin user groups [table 16]. Of the total cohort, 7217 patients started on biphasic insulin aspart ogld, of which 5995 (83.1%) were insulin nave and 1222 (16.9%) were insulin users . After 24 weeks of starting or switching to biphasic insulin aspart, hypoglycaemic events reduced from 0.2 events / patient - year to 0.0 events / patient - year in insulin nave group and from 2.2 events / patient - year to 0.1 events / patient - year in insulin users group . Body weight decreased and quality of life improved after 24 weeks of treatment [tables 5 and 6]. Biphasic insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [table 7]. Of the total cohort, 117 patients started on basal + insulin aspart ogld, of which 58 (49.6%) were insulin nave and 59 (50.4%) were insulin users . After 24 weeks of starting or switching to biphasic insulin aspart, hypoglycaemic events reduced from 2.7 events / patient - year to 0.6 events / patient - year in insulin nave group and from 4.9 events / patient - year to 1.4 events / patient - year in insulin users group . Body weight decreased and quality of life improved after 24 weeks [tables 8 and 9]. Basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to basal + insulin aspart oglds for both insulin nave and insulin user groups [table 10]. Of the total cohort, 1001 patients started on insulin detemir ogld, of which 865 (86.4%) were insulin nave and 136 (13.6%) were insulin users . After 24 weeks of starting or switching to biphasic insulin aspart, hypoglycaemic events reduced from 1.0 events / patient - year to 0.2 events / patient - year in insulin nave group and from 2.3 events / patient - year to 0.7 events / patient - year in insulin users group . A decrease in body weight and improvement in quality of life was observed at the end of the study [tables 11 and 12]. Insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir oglds for both insulin - nave and insulin user groups [table 13]. Of the total cohort, 734 patients started on insulin aspart ogld, of which 583 (79.4%) were insulin nave and 151 (20.6%) were insulin users . After 24 weeks of starting or switching to insulin aspart, overall hypoglycaemia decreased from 0.6 events / patient - year to 0.0 events / patient - year in insulin naive group and from 2.7 events / patient - year to 0.5 events / patient - year in insulin users group . A decrease in body weight and improvement in quality of life insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin aspart oglds for both insulin nave and insulin user groups [table 16]. Our study reports improved glycaemic control (hba1c, fpg, pppg) and quality of life following 24 weeks of treatment with any of the insulin analogues (biphasic insulin aspart; basal + insulin aspart; insulin detemir; insulin aspart) with or without ogld . Sadrs including major hypoglycaemic events or episodes did not occur in any of the study patients after 24 week of treatment . Though the findings are limited by number of patients, still the trend indicates that insulin analogues can be considered effective and possess a safe profile for treating type 2 diabetes in south india.
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The design of h2 oxidation and production electrocatalysts is critical for the development of alternative renewable energy technologies . The [fefe]-hydrogenase enzymes catalyze these reactions effectively with binuclear fe metal centers, as depicted in figure 1 . Fourier transform infrared spectroscopy (ftir) and electron paramagnetic resonance (epr) studies on the native enzyme have identified a mixed - valence oxidized state, hox, and a diamagnetic reduced state, hred . The hox state is well - characterized and features an obvious site for binding the h2 substrate on the distal fe center, denoted fed in figure 1 . The structure of the hred state is less clear because x - ray crystallography of biological systems cannot easily resolve hydrogen atoms . Although hypothesized for more than a decade, an azadithiolate bridgehead with an amine group pendant to the fe metal center has only recently been confirmed experimentally . Relative to biophysical studies, an advantage of model systems is that hydride and dihydrogen species are readily observed by h nmr and, in some cases, x - ray crystallography . Moreover, model systems are more computationally tractable, thereby providing additional structural and mechanistic insights . Oxidized (hox) and reduced (hred) intermediates of the [fefe]-hydrogenase enzyme s active site with the azadithiolate (adt) cofactor . A variety of h2-producing model systems inspired by [fefe]-hydrogenases have been synthesized . Many of these models suffer from high overpotentials (i.e., negative reduction potentials) for hydrogen production . The recent h2-producing models fe2(xdt)(co)2(dppv)2 (xdt = pdt, adt), depicted in figure 2, rely on an electrode in place of the [fe4s4] clusters that normally support electron transfer in the enzyme . Furthermore, in these models the cn and one of the co ligands are replaced by diphosphine (dppv) ligands . The presence of sterically crowded, electron - rich ligands on diiron carbonyl has been shown to stabilize the desired terminal hydride species . Through this approach, the most recent advance has revealed a key intermediate wherein the hydride is dihydrogen bonded to the protonated azadithiolate cofactor . This paper analyzes computationally the recent advances in the models and chemistry of the [fefe]-hydrogenases . This paper has two components: benchmarking the computational methodology for these model systems and providing insights into the mechanism of h2 production . The benchmarking provides the validation that is necessary to support the more hypothetical mechanistic predictions . In the first part of this paper, the computational methods are benchmarked by comparing experimental and theoretical structures, infrared (ir) vibrational frequencies of the co groups, reduction potentials, and relative pka values . In particular, we examine these model systems in the various oxidation states and protonation states along proposed mechanistic pathways for h2 production . Of particular interest, we aim to clarify the nature of the doubly protonated ammonium - hydride intermediate, which has been characterized crystallographically for this bimetallic system . Such doubly protonated intermediates have not been observed in bioinspired monometallic ni - based catalysts, although a mono - fe catalyst in the doubly protonated state has very recently been characterized by dubois and co - workers . We also present additional experimental data to validate the theoretical studies and to test predictions generated by the calculations . Ii, we introduce the nomenclature for the species studied and describe the computational methods used to calculate the structures, co vibrational frequencies, reduction potentials, and pka values for these species . Section iii presents the results and analysis, starting with the geometries and vibrational frequencies, followed by the reduction potentials and an analysis of the spin densities to determine the oxidation states of the metal centers . The second part of this section focuses on the protonation states and relative pka values and presents an analysis of possible h2 evolution mechanisms in the context of the experimental and computational data . Concluding remarks are presented in section iv . The diiron model systems can adopt numerous isomeric and geometric forms . Borrowing the nomenclature generally employed in enzymatic studies, the fe center with the hydride is denoted distal (fed) and the opposite fe center is denoted proximal (fep). Herein, we will abbreviate the (fepfed)(xdt)(co)2(dppv)2 catalysts as [(fefe)xdt] for notational simplicity . Figure 2 depicts the electrocatalyst in the terminal hydride (th) form with xdt representing either a propanedithiolate (pdt), [th-(fefe)pdt], or an azadithiolate (adt), [th-(fefe)adt]. In this notation, figure 3 depicts all of the protonation states of the [(fefe)adt] species studied in this work . A similar scheme for the [(fefe)pdt] species is available in the supporting information (figure s17). Model of the hred state of the enzyme, where the role of the [fe4s4] cluster is replaced by an electrode and the other ligands are replaced by 1,2-bis(diphenylphosphino)ethylene (dppv). The x represents the heteroatom in the dithiolate bridge (xdt = pdt, adt). Schematic depiction of the various [(fefe)adt] species, where each row represents a different protonation state of the catalyst . Only the isomers with the lowest free energies the dashed lines (p1, p2, p3, p4, and p5) correspond to pathways for which structural and electronic changes can be monitored by co values; they do not necessarily correspond to reaction pathways . Although the terminal hydride (th) species are thermodynamically less stable than their associated bridging hydride (h) isomers in model systems, experiments indicate that the terminal hydride species are much more catalytically active: they generate h2 faster and at less negative potentials than their bridging hydride counterparts . The dependence of the reduction potential on the regiochemistry of the hydride remains unexplained but appears to be catalytically relevant . Whereas the [(fefe)pdt] complex requires strong acids to form the terminal hydride species, [th-(fefe)pdt], the nearly isostructural [(fefe)adt] complex can be protonated with weak acids due to the proposed relay activity of the azadithiolate . Specifically, the presence of the azadithiolate bridge permits the formation of the ammonium tautomer, [(fefe)adt - h], and subsequent intramolecular proton transfer to the fed leads to the terminal hydride species, [th-(fefe)adt] (figure 3). Both [th-(fefe)adt] and [th-(fefe)pdt] convert to their respective bridging hydride isomers irreversibly at room temperature . Upon equilibration, two bridging hydride species form, sym-[h-(fefe)adt] and unsym-[h-(fefe)adt], which differ in the orientation of the dppv ring on the fed center relative to the dppv ring on the fep center (figure 3). The adt - containing hydride species can undergo a second protonation with stronger acids, leading to the doubly protonated species, [th-(fefe)adt - h] (figure 3). Again, [th-(fefe)adt - h] isomerizes into two bridging hydrides, sym-[h-(fefe)adt - h] and unsym-[h-(fefe)adt - h], albeit at a slower rate than the singly protonated analog . Although less thermodynamically stable than the bridging hydride species, the terminal hydride species, [th-(fefe)adt - h], is poised for h2 production due to the close proximity of the ammonium and fe - hydride centers in the doubly protonated species . Density functional theory (dft) calculations were performed to characterize the structural and energetic properties of the pertinent species in the catalytic scheme . All calculations were performed using the gaussian 09 electronic structure program . For geometry optimizations, the starting geometries were obtained from the corresponding crystal structures, which are available for the [(fefe)pdt], [(fefe)pdt],unsym-[h-(fefe)pdt], and unsym-[h-(fefe)pdt] species, as well as the [(fefe)adt] and [th-(fefe)adt - h] species . The starting geometries for species without crystal structures were obtained by manually altering the most closely related crystal structure . Optimizations in the gas phase and solution phase resulted in similar structures for several representative species, so gas phase optimizations were used herein for computational efficiency . A comparison of the structures optimized in the gas phase and solution phase for several representative species is available in the supporting information (table s2). The dft calculations were performed using the b3p86 density functional with the stuttgart pseudopotential and associated basis set of preuss and co - workers (sdd) for the fe centers, the 6 - 31 g * * basis set for the active hydrogens, the 6 - 31+g * basis set for the bf4 counterions, and the 6 - 31 g * basis set for all other atoms . To obtain the solvation free energies, we used the conductor - like polarizable continuum model (c - pcm) with the bondi atomic radii and included nonelectrostatic interactions, namely, the dispersion, repulsion, and cavitation energies . The solvation free energies were calculated in dichloromethane (ch2cl2) to coincide with the experiments unless otherwise stated . The co vibrational frequencies were calculated within the harmonic model and were scaled by a factor of 0.9850, as indicated by previous studies . The vibrational frequency shifts, co, were calculated for the dashed pathways shown in figure 3 . For the [th-(fefe)adt] and [th-(fefe)adt - h] species, the presence of the semibridging co ligand allows for the assignment of two distinguishable co stretching frequencies: the higher co corresponds to the terminal co attached to the fep center, and the lower co corresponds to the semibridging co. for all other species, both co ligands are terminal, so their stretching frequencies are less separated . In general, dft does not consistently provide reliable absolute pka s and reduction potentials because of limitations in the functionals, basis sets, and solvation models, as well as uncertainties in the free energies of the electron, proton, and electrode . As discussed previously, the calculation of pka s and reduction potentials relative to related reference systems with known experimental values has been shown to be more reliable because many of the systematic errors cancel . For this reason, we discuss only relative reduction potentials and pka s in this paper . The structures and energies of all complexes discussed herein are provided in the supporting information . The reaction free energies associated with reduction and deprotonation were calculated using the born haber thermodynamic cycle . The reaction free energy for reduction, gr(e), was calculated using the expression1 in this expression, gs(red) and gs(ox) are the solvation free energies of the reduced and oxidized species, respectively, and ggas is the gas phase reaction free energy calculated using the standard relation ggas = hgas tsgas. A similar expression was used to calculate the reaction free energy for deprotonation, gr(h). For reasons mentioned above, the reduction potentials were calculated relative to a related reference reaction for which the experimental value is known . This procedure accounts for systematic errors associated with the selected theoretical method and basis set and avoids the determination of the free energies of the electron and the electrode because these terms cancel . Detailed descriptions of this methodology can be found elsewhere . For our specific application, we used the following expression to calculate the reduction potentials:2here, e is the reduction potential of the [(fefe)adt] species of interest, eref is the experimentally measured reduction potential of the reference species, which is the [(fefe)pdt] species in this work, gr(e) is the calculated difference in reaction free energies (eq 1) for reduction of the species of interest and the reference, and f is the faraday constant . All calculated and experimental reduction potentials are given in volts (v) relative to the ferrocene / ferrocenium (fc / fc) couple in ch2cl2 . The irreversible reduction potentials of the doubly protonated species are calculated relative to the reduction potentials of the corresponding singly protonated species and are labeled eprot. The cyclic voltammetry (cv) experiments were performed at 273 k, and the reduction potentials were also calculated at this temperature . We used the standard relationship, pka = gr(h)/(rt ln 10), to calculate the pka values . In this expression, gr(h) corresponds to the calculated reaction free energy for deprotonation, r is the gas constant, and t = 233.15 k to coincide with experimental conditions . Due to the limited experimental pka data for the [(fefe)pdt] systems and its inherent inability to doubly protonate, these complexes were not used as references . Instead, we calculated pka, which is defined to be the difference between the pka values for two specified states of the [(fefe)adt] complex . The reported pka values were calculated in ch2cl2 to coincide with experimental conditions and in acetonitrile (ch3cn) to coincide with the reference pka values used in the interpretation of the experimental values because a pka scale is not well established for ch2cl2 . We optimized the geometries of the various species discussed above and compared the structures and key vibrational frequencies to available experimental data . The optimized geometries are in reasonable agreement with the x - ray crystal structures . Table 1 provides a comparison of the most relevant bond lengths for the various species . Structural and energetic information on all systems studied herein are provided in the supporting information . The relative free energies of the species depicted in figure 3 are provided in table 2 . Moreover, the relative free energies of these species and other isomers of higher free energy are provided in figures s15 and s16, supporting information . Experimental data from ref (40). Experimental data from ref (46). Experimental data from ref (35). Experimental data from ref (41). Experimental data from ref (38). Not applicable because the hydrogen is not present for certain species or the distance is not relevant for the doubly protonated species . Values given in kcal / mol relative to the unprotonated species, [(fefe)adt], in ch2cl2 at 298.15 k. the free energy of a solvated proton should be included to obtain free energies relevant to the protonation reactions . The optimized geometry for the doubly protonated species, [th-(fefe)adt - h], exhibited one key difference from the crystal structure, namely, the dihydrogen distance nhhfe . This dihydrogen distance was dhh = 1.88 in the crystal structure and dhh = 1.40 in the optimized geometry . Thus, dft leads to a significantly shorter dihydrogen distance that corresponds to a stronger dihydrogen bond . A previous dft study on a model of the [fefe]-hydrogenase active site also showed an underestimation of the dihydrogen distance for a related doubly protonated species . This smaller dihydrogen distance in the [th-(fefe)adt - h] species was found for a wide range of density functionals, as shown in table s3, supporting information . However, dihydrogen bond distances have also been shown to be extremely sensitive to weak intra- and intermolecular interactions, such as ion - pairing . Moreover, neutron diffraction experiments are expected to provide more reliable positions for hydrogen atoms than x - ray diffraction, which most likely overestimates this distance . A recent neutron diffraction study on a monometallic fe hydrogenase model using the noncoordinating counterion, bar4, found the nhhfe dihydrogen bond to be 1.49 . In contrast, the doubly protonated structure, [th-(fefe)adt - h], was determined with x - ray crystallography and included bf4 ions that could exhibit significant ion - pairing effects . Given the difference in the crystallographic and calculated dihydrogen bond length in [th-(fefe)adt - h], we hypothesized that this discrepancy was due to the omission of the bf4 ions in the geometry optimizations . To test this hypothesis, we also optimized the structure including one or two of the bf4 ions closest to the azadithiolate bridge in the crystal structure . Figure 4 illustrates that the geometry optimizations including either one or two bf4 ions improved the agreement between dft and experiment by shifting the amine bridgehead and elongating the dihydrogen distance . The bond lengths given in table 3 show that including the ion - pairing interactions preserves the fefe and feh distances while elongating the dihydrogen distance . Although the inclusion of counterions improves the agreement with the doubly protonated crystal structure, we omit the counterions in the calculations of other quantities, such as reduction potentials and spin densities, to maintain consistency among the various species studied under a range of experimental conditions . This structure was optimized in solution (ch2cl2) using c - pcm without any bf4 counterions . Superimposed structures of the doubly protonated species, [th-(fefe)adt - h], illustrating the better agreement between the crystal structure and the optimized geometries when bf4 ions are included in the calculations . The phenyl groups have been removed, and the fehhn interaction is circled for clarity . Color scheme: crystal structure (gray) and optimized geometry with no bf4 (red), one bf4 (blue), and two bf4 (green) counterions . The strong, distinctive absorptions at 2000 cm associated with co stretching can be used to probe changes in the electronic structure due to protonation, isomerization, reduction, and oxidation . We calculated the change in the co frequencies, co, for the five protonation reactions depicted by dashed lines in figure 3 . As shown in table 4, the calculated co values due to protonation are within 10 cm of the experimentally measured values . This level of agreement provides validation for the structures of species for which crystal structures are not available . Typically dft calculations are more reliable for changes in frequencies than for absolute frequencies; a comparison between the calculated and experimental absolute frequencies is provided in table s4, supporting information . For the present study, the previously reported spectroscopic studies were repeated using the acid [h(oet2)2]bar4 to eliminate the possibility of ion pairing arising from the coordination of bf4 when using hbf4et2o, which has been shown to participate in hydrogen - bonding interactions, particularly in complexes with protonated adt ligands . The agreement between calculated and experimental frequencies is better for the bar4 salts; these spectra are provided in the supporting information (figures s6 and s7). The p1, p2, p3, p4, and p5 protonation reactions are defined in figure 3 . The second co value is assigned to the semibridging co. the co values reflect the protonation sites, as indicated by both experimental measurements and theoretical calculations . The co values associated with n - protonation are typically characterized by co frequency increases of 20 cm . The experimental and calculated co values associated with n - protonation to form either the singly or doubly protonated species (p1, p4, and p5 protonations) exhibit this shift . While n - protonation is associated with a relatively small co shift, formation of the terminal and bridging hydrides (p2 and p3 protonations, respectively) is associated with much higher shifts of 70 cm . In both cases, two well - resolved bands are observed in the spectra, but the bands are much more energetically separated for the terminal hydride species than for the bridging hydride species . The band at lower frequencies in the terminal hydride species is assigned to the semibridging co, which exhibits a smaller co shift of 40 cm . Our calculated co values for the protonations leading to the terminal and bridging hydride species (p2 and p3 protonations, respectively) demonstrate this trend and agree well with the experimental values (table 4). An analogous figure and table for the [(fefe)pdt] complexes is provided in the supporting information (figure s17 and table s1). The calculated reduction potentials for the species in ch2cl2 are presented in table 5 . Because the [(fefe)pdt] species are used as the reference systems, these reduction potentials agree exactly with experiment by construction, and all other reduction potentials are calculated relative to these values . The agreement between theory and experiment for the [(fefe)adt] species is reasonable for the singly protonated species with errors of 0.05 v vs fc / fc in ch2cl2 . Additional calculated reduction potentials are provided in the supporting information (table s5). A detailed analysis of the metal oxidation states of the reduced species in terms of the spin densities is provided in the next subsection . Unless otherwise indicated . [(fefe)pdt] reactions were used as the reference reactions, so the calculated and experimental values agree by construction . Experimental data from ref (79). Quasi - reversible reaction, so ep is reported . E = 1.82 v vs fc / fc in ch2cl2 if calculated using the symmetric bridging form . Experimental data from ref (38). Irreversible reaction, so ep is reported . E = 1.83 v vs fc / fc in ch2cl2 if calculated using the symmetric bridging form . Due to difficulties in determining a suitable reference system for the doubly protonated species, we calculated the difference between the reduction potentials for the doubly and singly protonated species (eprot). Using this formulation, we calculated eprot = 0.59 v vs fc / fc in ch2cl2 . As expected from basic electrostatic arguments, the doubly protonated species has a less negative reduction potential than the singly protonated species . The experimentally measured difference in these reduction potentials was 0.39 v vs fc / fc in ch2cl2 . As discussed above, this error could be due to the neglect of bf4 in the calculations . Note that bf4 ions were present in the electrochemical measurements for the doubly protonated species but not for the singly protonated species . Furthermore, the effects of ion pairing on reduction potentials are known to be pronounced in solvents of low dielectric constants, such as ch2cl2 . Thus, the difference in the counterions present for the electrochemical experiments of the singly and doubly protonated species could lead to discrepancies in the comparison between calculated and experimental results . Note that the experimentally measured reduction potentials for the terminal hydride species are for quasireversible couples, which could introduce additional discrepancies . For species with an unpaired electron, we analyzed the spin densities to determine the localization of the unpaired electron . For the reduced terminal hydride species, [th-(fefe)adt], the unpaired spin density is localized entirely on the fep center, suggesting that the fep center is reduced . This localization on the fep center is similar for the reduced terminal hydride species, [th-(fefe)pdt], which is produced at a similar potential as the [th-(fefe)adt] species, indicating that the dithiolate is not a major influence on the redox behavior for the terminal hydride species . The spin densities pertaining to the [(fefe)pdt] complexes are provided in the supporting information (table s6). Previous studies also examined the metal oxidation states upon reduction of [h-(fefe)pdt] by analyzing the spin density with the bp86 functional and epr spectra of the mixed - valence species . These studies showed that reduction of sym-[h-(fefe)pdt] resulted in a species with the unpaired electron delocalized between the fep and fed centers, sym-[h-(fefe)pdt]. However, reduction of unsym-[h-(fefe)pdt] resulted in a species with the unpaired electron localized on the fep center, unsym-[h-(fefe)pdt]. Our calculations revealed a similar trend with the bridging hydride species of the singly and doubly protonated [(fefe)adt] (table 6), although several important differences were observed . In the singly protonated symmetric and unsymmetric [(fefe)adt] bridging hydride species, sym-[h-(fefe)adt] and unsym-[h-(fefe)adt], the orientations of the dppv ligands on the two fe centers furthermore, the hydride is located symmetrically between the two fe centers in both isomers for the oxidized state ., the bridging hydride is perturbed from this symmetric position . In the reduced unsymmetric bridging hydride species, the hydride is more closely bound to fed (dfeh = 1.58) than to fep (dfeh = 2.05) and resembles a terminal hydride . In the reduced symmetric bridging hydride species, the hydride is more symmetrically bound to the two iron centers (dfeh = 1.60 for fed and dfeh = 1.77 for fep). The observation that the reduction of sym-[h-(fefe)adt] maintains a more symmetric bridging hydride is consistent with the observation that the spin density is somewhat delocalized between the two fe centers . In contrast, reduction of unsym-[h-(fefe)adt] leads to a much more asymmetric bridged hydride, and the spin density is localized on fep, which does not have a bound hydride . Thus, unsym-[h-(fefe)adt] is similar to [th-(fefe)adt] in that reduction occurs at the fep site, but reduction of unsym-[h-(fefe)adt] also involves motion of the bridged hydride toward the fed site to resemble a terminal hydride . These observations are consistent with the observation that the terminal hydride species is reduced more easily (i.e., at a slightly less negative potential) than the unsymmetric bridging hydride species (table 5). The bond lengths given without parentheses correspond to the oxidized species as drawn, while those given in parentheses correspond to the reduced mixed - valence species (not drawn). The spin densities of the reduced mixed - valence species are reported in table 6 . The presence of the amine group in the azadithiolate bridgehead affects the degree of asymmetry observed upon reduction of the symmetric bridging hydride species . Although the bridging hydride is more symmetric in the sym-[h-(fefe)adt] species than in the unsym-[h-(fefe)adt] species, more asymmetry is exhibited in the sym-[h-(fefe)adt] than in the analogous sym-[h-(fefe)pdt] species . In the previous study of [(fefe)pdt] bridging hydride species, reduction of sym-[h-(fefe)pdt] preserves the symmetry of the hydride position to a higher degree, with dfeh = 1.68 and dfeh = 1.70 for the fep and fed centers, respectively . We have confirmed these observations using the level of theory described within the present paper and various other basis sets . Moreover, these nearly symmetric feh bond lengths are associated with nearly equal spin densities on the two fe centers . As shown in figure 5 and table 6, the reduced symmetric protonated [(fefe)adt] system, sym-[h-(fefe)adt], exhibits more asymmetry in these feh bond lengths and unequal delocalized spin densities on the two fe centers . The reduction of the doubly protonated bridging hydride species, unsym-[h-(fefe)adt - h] and sym-[h-(fefe)adt - h], follows a similar trend but displays a slightly smaller degree of delocalization of the spin density, as shown in table 6 . According to the distances given in figure 5, the asymmetry of the bridging hydride is slightly more pronounced in both the symmetric and unsymmetric forms for the doubly protonated species . Thus, reduction is expected to occur primarily at the nonhydride fep center, as supported by the unpaired spin densities given in table 6 . This pattern for the electron localization is also seen in doubly protonated terminal hydride species, which will be discussed below . Previous experiments on the [(fefe)adt] system estimated a small difference in the pka of the first and second protonation steps (pka 2); however, our calculated values indicated much larger differences, as seen from pka(p4 this discrepancy between experiment and theory prompted a reinvestigation of the acid base chemistry of the [(fefe)adt] species . The pka values can be estimated by nmr measurements because each protonated species displays a unique p nmr spectrum . The experimental details and spectra are provided in the supporting information (figures s12 and s13). According to these experiments, the protonation reactions leading to the formation of [(fefe)adt - h], [th-(fefe)adt], and [th-(fefe)adt - h], defined as p1, p2, and p4 in figure 3, correspond to pka values of> 14.2, 15.5, and 6, respectively . The protonation pathways p1, p2, p3, and p4 are defined in figure 3, and the pka is determined for the protonated species in each case . These experiments were performed in ch2cl2 solvent, but the reference pka values pertain to ch3cn solvent . For reasons discussed in the methods section this procedure accounts for systematic errors associated with the selected theoretical method and avoids the determination of the free energy of the solvated proton because this term cancels . Note that the experimentally measured values are only qualitatively meaningful because the experiments were performed in ch2cl2, which does not have a well - defined pka scale, and the reference pka values of the acids pertain to ch3cn . As a result, we calculated the relative pka values in both ch2cl2 and ch3cn . Additionally, the effects of homoconjugation in these experiments may distort the apparent pka measured . As discussed above in the context of comparing the crystal structure and optimized geometries furthermore, it has been shown experimentally that ion pairing between the bf4 counterion and the ammonium center can shift the equilibrium of the singly protonated species toward [(fefe)adt - h]. P1) value indicates that the fed site is more basic than the amine by at least 1.3 pka units; however, the amine is more readily protonated than the stereochemically crowded fe site . The greater basicity of the fed site than the amine is corroborated by our calculated pka(p2 p2) value measured experimentally indicates that n - protonation of [th-(fefe)adt] to form [th-(fefe)adt - h] requires much stronger acids, as supported by our calculated large, negative pka(p4 again, it is worth noting that the presence of the bf4 ions in the experiments may contribute to the remaining discrepancy between theory and experiment . Moreover, the theoretical calculation of pka differences for species with different charges (i.e., + 2 and + 1 for the doubly and singly protonated species) is more challenging . Turning our attention to the bridging hydride isomers, previous experiments have suggested that the conversion to the bridging hydride isomers is irreversible, and the bridging hydride is estimated to be at least 2 pka units less acidic than the terminal hydride . This observation is supported by the large, positive calculated pka (p3p2) value and the thermodynamic stability of the bridging hydride isomers . These pka trends are reproducible using different functionals, as indicated by additional data provided in the supporting information (table s8). The proposed mechanisms for hydrogen evolution catalyzed by the [(fefe)adt] system for weak and strong acids are depicted in figure 6 . Initially, the amine group on [(fefe)adt] is protonated to form the ammonium tautomer, [(fefe)adt - h]. An intramolecular proton transfer occurs from the n to the fed to form the terminal hydride, [th-(fefe)adt]. For weak acids, the terminal hydride species, [th-(fefe)adt], this pathway is shown in dotted lines in figure 6 . For stronger acids, the intramolecular proton transfer is followed by another protonation at the amine group to generate [th-(fefe)adt - h]. A possible mechanism for this process is indicated with solid lines on the right side of figure 6 . The values for the spin densities and bond distances for the species associated with this pathway are given in table 8 . The steps indicated with solid lines along the perimeter represent the proposed pathway discussed in the main text . Our calculations indicate that reduction of [th-(fefe)adt - h] leads to the mixed - valence complex, [th-(fefe)adt - h]. The spin densities and bond distances of this species are given in table 8 . This mixed - valence species is characterized by the unpaired electron localized on the fep center, which is in agreement with the reduction scheme exhibited by the [th-(fefe)adt] and [th-(fefe)pdt] species discussed previously . The nhhfe distance is shorter in [th-(fefe)adt - h] (dhh = 1.30) than in [th-(fefe)adt - h] (dhh = 1.40) but is still larger than the distance in molecular h2 (dhh = 0.74). Although we were unable to locate a minimum - energy species with h2 weakly bound to an fed(i) center, we found another type of h2 adduct intermediate with the unpaired electron delocalized between the fep and fed metal centers . This h2 adduct, [h2-(fefe)adt], is higher in free energy than the mixed - valence doubly protonated species, [th-(fefe)adt - h], by 5.82 kcal / mol but has several features that are potentially catalytically relevant, as will be discussed below . Note that previous theoretical studies on small models of the [fefe]-hydrogenase active site found that reduction of a doubly protonated fep(ii)fed(ii) species could lead to the formation of a stable mixed - valence h2 adduct intermediate, fep(i)fed(ii), where the fep center is the site of reduction and the h2 ligand is loosely bound to the fed(ii) center . These mixed - valence intermediates are similar to the [th-(fefe)adt - h] species found herein; however, the [h2-(fefe)adt] species was not found in these previous studies . In contrast to the [th-(fefe)adt - h] species, the [h2-(fefe)adt] species features a much shorter nhhfe distance (dhh = 0.79), suggesting an activated h2 molecule . In conjunction with the activated h2 bond distance, the average distance between each hydrogen and the fed center is elongated (dfeh = 1.75), suggesting that the h2 ligand is weakly bound to the fed center and poised for release . Another important structural feature is that unlike all terminal hydride species reported in the present paper, the semibridging co ligand in this h2 adduct is nearly symmetrically bridged between the fep and fed centers, where dfeco= 2.06 and 1.89, respectively . The fepfed distance contracts (dfefe = 2.69) to accommodate this bridging interaction . This structural property is reminiscent of the enzyme s hox state, which features a nearly symmetrically bridged co ligand (figure 1). Moreover, the spin density is now delocalized between the fep and fed center, 0.43 and 0.45, respectively, warranting the [h2-(fefe)adt] assignment . This delocalization of the spin density suggests the possibility for intramolecular electron transfer to occur between the fep and fed centers concomitant with formation of a shorter h2 bond . Note that the proposed mechanism in figure 6 is similar to mechanisms proposed previously for [fefe]-hydrogenases in terms of the reduction and protonation steps . Our objective is to characterize the proposed intermediates and to analyze aspects such as dihydrogen bonding and electron delocalization or localization with respect to the metal centers . Release of h2 from [h2-(fefe)adt] would generate another mixed - valence state, the [(fefe)adt] species shown in figure 6 . This mixed - valence species is closely related to the hox state of the enzyme shown in figure 1 . Interestingly, the spin densities for this species, which are given in table 8, indicate fe oxidation states that are opposite to the various other mixed - valence species discussed in this paper . Whereas the spin density was localized on the fep center for the other purely mixed - valence species, such as [th-(fefe)adt] and [th-(fefe)adt - h], this hox - like species has the spin density entirely localized on the fed center, suggesting that the fed center is now more reduced than the fep center . Upon formation of this [(fefe)adt] species, the bridging co this analysis of the mechanism points to the importance of the bridging co ligand, which seemingly behaves as a lever and indicator for intramolecular electron transfer between the fe centers . Moreover, there is 0.16 spin density localized on the symmetric bridging co ligand in the [h2-(fefe)adt] species, suggesting that it may be even more involved in the electron transfer process . In this paper, we investigated a proton reduction catalyst, [(fefe)adt], which is modeled after the [fefe]-hydrogenase enzyme . This model is characterized by incorporation of the azadithiolate bridge, which facilitates the formation of a doubly protonated species through a proton relay . The calculated results agree well with the experimental data for the geometries, co vibrational stretching frequencies, and reduction potentials for the systems studied . The calculations illustrate that the nhhfe dihydrogen bonding distance in the doubly protonated species is highly sensitive to the effects of ion - pairing between the ammonium and bf4 counterions, which are present in the crystal structure . Specifically, the inclusion of bf4 counterions leads to a significantly longer dihydrogen bond . Comparable weak interactions with the protein backbone might be expected to influence the hh bonding in the enzyme . Analyses of spin densities provide insight into the oxidation states of the metal centers for the reduced singly and doubly protonated species . The non - hydride fe center (fep) was found to be the site of reduction for terminal hydride species and for unsymmetric bridging hydride species . In contrast, the reduced symmetric bridging hydride species exhibited a significant degree of spin delocalization between the fe centers, although these species exhibited more asymmetry than the analogous [(fefe)pdt] species . These differences in the degree of spin localization, as well as the movement of the hydride upon reduction of the unsymmetric bridging species, may be related to the experimental observation that the terminal hydride species are reduced at less negative potentials than their bridging hydride isomers . This analysis suggests that the bridging hydrides may be considered to be quasi - terminal hydrides for the unsymmetric species . A combined experimental and theoretical study of the relative pka values for the various species in the catalytic cycle provides further insights . The fed site was found to be more basic than the amine, although the amine is more readily protonated . Moreover, the bridging hydride species was found to be more thermodynamically stable than the terminal hydride species . These observations are consistent with the initial protonation of the amine, followed by intramolecular proton transfer to the fed site to produce a terminal hydride species, followed by isomerization to the bridging hydride species at room temperature but not at low temperatures . In terms of forming the doubly protonated species, the amine site is significantly less basic for the terminal hydride species than for the unprotonated species . Both the experimental and theoretical data indicate that a stronger acid is required for this second protonation . In addition, these calculations implicate a possible pathway for h2 evolution that involves an intermediate with h2 weakly bonded to one fe, a short h2 distance similar to the molecular bond length, the spin density delocalized over the two fe centers, and a nearly symmetrically bridged co ligand . H2 bonding is relatively weak and that the product h2 is poised for release . Upon h2 release, the mixed - valence species produced is closely related to the hox state of the enzyme . The key features of this proposed mechanism highlight the roles of the ammonium - hydride interaction, flexibility of the bridging co ligand, and intramolecular electron transfer between the fe centers in the catalytic cycle . While these bioinspired models are effective catalysts for h2 production, the challenge of designing molecular electrocatalysts that can perform with weaker acids and lower overpotentials remains . Further investigation of the effects of ligand substitution at the fe centers will help guide the design of more effective h2-producing catalysts.
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Clinical epidemiological research focuses on patient - oriented studies of risk factors, screening, diagnostics, treatment, and prognosis . Prevention and treatment of disease are the overarching goals of health research and thus the ultimate goal of our academic activity.13 it is important to keep this in mind, as academic success at an individual level is often measured solely in terms of publishing original peer - reviewed papers, defined by many as the coin of the realm.4 the selection and training of future researchers are decisive factors in the quality of research . Currently, we face a shortage of talented researchers in clinical medicine to take up future challenges.48 large amounts of money are invested in research, and there are strict requirements for the research process with regard to preparing funding applications and operational protocols . However, the literature provides little information on the characteristics of successful students in phd and postdoctoral programs . This information is needed to identify candidates who will bring the greatest value to the research enterprise at individual universities and hospitals and ultimately to patients . A number of quantitative criteria are available to guide the selection of more senior researchers for open positions . These include number of papers published, position in author sequences, numbers of citations, and the h - index, although the relation between these criteria and the quality of research is debatable.9 as this type of information is not available for selecting phd students and postdoctoral candidates, it is necessary to rely on grades from previous education, performance in earlier employment, strength of an application, and sometimes an interview . These characteristics are not always accurate indicators of success in graduate and postgraduate education . In medical school, for instance, excellent grades are often achieved through highly individualized study . Grades are not necessarily valuable in predicting which students will become successful in conducting research, as it requires a far wider range of competencies . The training of clinical researchers is organized differently from one country to another, often based on tradition . In some european countries, many clinical researchers have a doctoral degree, while in the usa they may hold a master s degree.10 similarly, postdoctoral work may be conducted solely as part of a research appointment or divided between research and clinical work in a clinical research appointment.8 the principles discussed in this perspectives paper apply to diverse courses of training . More than 10 years ago, after having supervised a number of phd students, i became interested in the factors characterizing those who completed their phd programs in time, published papers in recognized peer - reviewed international journals, and experienced a clear improvement in their research skills during their training . In recent years, this interest has grown to include identifying which doctoral candidates on the threshold of a postdoctoral program would develop into independent researchers with innovative research agendas, with the aptitude for establishing their own team of researchers, and with the ability to finance their own research.8 an essential criterion for success as a senior researcher is the ability to obtain support for their own research from peer - reviewed funding agencies . At the beginning of postdoctoral training, a clear picture starts to emerge of which postdoctorals formulate innovative research questions and which primarily conduct confirmative research . The challenge is to select individuals with talents and personal qualities that allow them to develop the skills of independent and resourceful researchers . Through acquisition of knowledge and honing of skills it is well documented that expert professional performance rests on knowledge and skills that cannot be acquired solely through teaching and individual study . The key requirement is personal commitment in addition to research experience.11,12 in this perspectives paper, i report some simple characteristics that i have identified as determinants of success in a phd program and subsequent independent research in a postdoctoral program . My conclusions derive from personal experience both retrospective mainly cross - sectional observations and more prospective cohort - oriented assessments . I started by jotting down some notes and then began to work systematically on principles for selecting phd students and postdoctorals . Rather, it may provide a qualitative description of a screening process and checklist . Based on my material (that is, the individuals we have identified as potential candidates), i have calculated some simple predictive values for individual variables . Serendipitously, many of the words i found to describe the successful researcher at the beginning of a career began with the letter i . The recruitment of phd students varies considerably from one institution to another and from one country to another . In some cases, it is an ad hoc procedure, in which potential phd students contact desired supervisors . In other cases, phd programs have two overall objectives: to extend knowledge about an important and as yet unclarified health topic via a research project . This project ranges from basic research, such as testing specific hypotheses related to mechanisms of disease, to more applied clinically oriented research, that is, studies of risk factors, diagnostic approaches, treatment and rehabilitation, and health services.13to provide extensive training to improve the phd student s competencies, through learning research methods and interaction with the research supervisor, other students, administrative and other staff, funding agencies, journal editors, teaching staff, and in some cases patients.14 successful interaction requires initiative, resourcefulness, skills in communication and collaboration, and an approachable manner (both on the part of the student and the supervisor). To extend knowledge about an important and as yet unclarified health topic via a research project this project ranges from basic research, such as testing specific hypotheses related to mechanisms of disease, to more applied clinically oriented research, that is, studies of risk factors, diagnostic approaches, treatment and rehabilitation, and health services.13 to provide extensive training to improve the phd student s competencies, through learning research methods and interaction with the research supervisor, other students, administrative and other staff, funding agencies, journal editors, teaching staff, and in some cases patients.14 successful interaction requires initiative, resourcefulness, skills in communication and collaboration, and an approachable manner (both on the part of the student and the supervisor). This requires the goals and research plan for the project, as well as the teaching curriculum, to be well defined . In addition, to successfully complete a phd research program, the student and the supervisor must possess a certain positive psychological energy as a driving force, which includes both motivation and enthusiasm . The following elements the content of the project and teaching, positive energy in the student and supervisor, and opportunities for wide - ranging interaction all need to be present in the phd training . In addition, independent and individual learning is an important element in a phd program, which comes as a surprise to many phd students.15 it is critical to the phd outcome that the research project be sound ., of clinical and public health relevance,16 and sufficient funding must be available covering both project costs and the student s compensation (figure 1). A good early predictor of success in a phd program is the student s assumption of responsibility for preparing funding applications and seeking administrative support and advice . Many supervisors have difficulty finding sufficient time for supervision and for familiarizing themselves with the subject content of a specific research area and make demands . In my experience, problems in successfully completing a phd program stem 1) from a poorly planned and inadequately planned project, 2) from clinical supervisors who are insufficiently aware of their own level of competency (eg, in their grasp of research methodology) and who do not have enough time for supervision, and 3) from supervisors with insufficient clinical knowledge or a lack of understanding of the framework for clinical research, despite their expertise in methodology . It is not always clear at the beginning of a phd program that a student lacks the qualifications necessary to undertake research on a given subject . Early signs that the process is not going smoothly are student ambivalence, lack of motivation, or when a student comes to the department infrequently or cancels meetings . They may be sensitive to criticism or feel they are highly knowledgeable . Despite their intelligence a student s performance can also be impacted by personal challenges, such as family problems or a change in a spouse s employment . Supervisors and chairs in a good research department should promote the following qualities: a good working environment, in which management clearly communicates the department s vision, mission, and values particularly the soft values such as honesty, communication, support, respect, fairness, predictability, competence, and aspirations.responsibility, demonstrated by behaving positively, holding to agreements, preparing carefully for meetings, arriving on time, observing deadlines, being result - oriented, respecting all groups of staff, collaboratively securing resources in the research environment, making an extra effort to use resources conscientiously, and maintaining high ethical standards.collaboration, achieved by actively sharing knowledge and information, giving constructive and honest criticism, contributing to solutions and improvements, and especially allowing time for students to solve problems themselves . A supervisor must articulate professional questions and resolve dilemmas to ensure progress in the research . A good working environment, in which management clearly communicates the department s vision, mission, and values particularly the soft values such as honesty, communication, support, respect, fairness, predictability, competence, and aspirations . Responsibility, demonstrated by behaving positively, holding to agreements, preparing carefully for meetings, arriving on time, observing deadlines, being result - oriented, respecting all groups of staff, collaboratively securing resources in the research environment, making an extra effort to use resources conscientiously, and maintaining high ethical standards . Collaboration, achieved by actively sharing knowledge and information, giving constructive and honest criticism, contributing to solutions and improvements, and especially allowing time for students to solve problems themselves . A supervisor must articulate professional questions and resolve dilemmas to ensure progress in the research . An atmosphere of institutional and individual competition is present in many research environments, produced by the great competition for research funding and appointments . Several different professional groups may be employed in a department, each with its own professional identity, and persons of different nationalities may converse in their own languages . In our program, at least 20%25% of phd students run into some kind of problems in the course of their phd program . Approximately 40%45% deliver a quality phd dissertation based on quality papers . Only ~5%10% of phd students, however, ultimately become independent researchers with their own research teams . In many cases, the reasons for the problems are not just the phd students qualifications but also include poorly planned projects and poor supervisors . In a number of cases, the project runs short of funds . These data correspond to data from the wellcome trust and cancer research, uk, that showed that among clinical phd graduates, a third progressed to a formal academic post . The british heart association has reported that only 40% of clinicians who completed a phd continued to be active in research.5 if a student enters a phd program from necessity or coercion (for instance, as a qualification for promotion or a career), the prognosis is far less favorable than when the driving force is a genuine interest in research . Early on, it is important to clearly identify the incentive for undertaking a phd program . Personally, i always set a 3-month probation period before a phd student is accepted, to clarify whether it was the right choice, both for the phd student and for the department . A firm agreement about a 3-month probation period does not result in a loss of face for a student who does not progress to final enrollment . I - determinants, mentioned previously and discussed in detail subsequently, are present . In selecting phd students, it is important to be aware of the wide range of components subsumed under the concept of competencies: personal characteristics, talent, knowledge, skills, and behavior . While the individual basis (personal characteristics and talent) is not easily changed, it is possible for a supervisor to influence the last three elements.12 positive personal characteristics and talent are, however, the factors that distinguish top researchers from average researchers . In addition, successful researchers are characterized by having clear goals and a research agenda.4,8 they also benefit from a committed mentor10,17,18 with deep insight about a subject, who is capable of giving qualified and independent advice without conflict of interest . They identify with their profession and understand the dynamics and funding climate of a research department . As mentioned previously, over the years i have recorded a number of characteristics that are indicative of a promising phd student . By chance, i have adjusted my list to make it fit the i - determinants and to promote understanding and dissemination . Although not exhaustive, the following terms describe the successful phd student (figure 2): interest in the phd program: this interest must be the essential motivation for seeking to obtain a doctorate, preferably combined with a professional need to obtain the degree . This i - determinant is a key marker for progression in the program and acquisition of knowledge and skills . Prior research experience (for instance, as a medical student) with peer - reviewed publications is also an excellent predictor.19incentive: potential candidates fall between two extremes regarding incentive . If this is combined with a genuine interest, it is a good predictor of success . At the same time this is also true if a candidate wishes to obtain a break from the clinical routine, to avoid other responsibilities, or to avoid a transfer to another city . Both the concepts interest and incentive are closely related to motives . In psychology, several types of motives have been distinguished . Internal or intrinsic (also called genuine) motives are related to the activities themselves.20 for example, a phd student is motivated to create important insight and knowledge . This will often include hard work and struggle to reach the desired outcome that is intrinsically connected to the activity itself . In contrast to internal motives are the so - called instrumental motives, where the motive bears no intrinsic relation to the activity itself . Some instrumental motives could be that the phd student wants to get promoted, get a specialist position, or a good salary . Internal motivation predicts positive outcomes, while multiple motives may have negative impact in an educational context.20idea: if the candidate has a good idea of what he or she wants to investigate, has sought out, and read a selection of the literature on the question to be researched, and if the idea is realistic, the probability of success is good . If, in addition, the idea is focused, the prognosis is better than if the idea is broad and diffuse.initiative: if the student shows initiative, is always well prepared, drafts proposals, and meets deadlines, the probability of success is far more likely than for a reactive student who expects proposals to be provided by a supervisor who always has to take the initiative . The prognosis is, as expected, far better for proactive personalities than for reactive personalities.integrity: personality plays an essential part in interactions with others . Personal characteristics predictive of success in a phd program are responsibility and realistic self - confidence . The prognosis is good for students with their own clearly grounded approaches to research, the research project, and the supervisor . The prognosis is also good for students who take responsibility for themselves and their programs and understand that obstacles and challenges help to drive personal development in the research process . The role of supervision should be very clearly defined as a professional relationship rather than a tutorial friendship.15 this commentary does not cover supervision, which is an independent discipline.15,21 clear communication is important in the relationship between supervisor and student . Expectations must match, and a framework must be agreed upon for the supervisory process . As stated earlier, it is important for a supervisor to have the right interest, competencies, and knowledge in relation to a given phd project . At the same time, the student s personal integrity is a prerequisite for the necessary discipline and time management (a major problem for many). Some universities issue formal contracts between the institution and phd students.interpersonal relationships: the ability to communicate well and share knowledge plays a major role in successful interaction with a supervisor, other phd students, and employees in the department . During interviews, i try to uncover the ability to maintain and develop positive interpersonal relationships, relying on the following words as signals: motivation, willpower, sensitivity, expectations, psychological balance, loyalty, frequency and duration of previous positions, and earlier conflicts . Interest in the phd program: this interest must be the essential motivation for seeking to obtain a doctorate, preferably combined with a professional need to obtain the degree . This i - determinant is a key marker for progression in the program and acquisition of knowledge and skills . Prior research experience (for instance, as a medical student) with peer - reviewed publications is also an excellent predictor.19 incentive: potential candidates fall between two extremes regarding incentive . If this is combined with a genuine interest, it is a good predictor of success . At the same time this is also true if a candidate wishes to obtain a break from the clinical routine, to avoid other responsibilities, or to avoid a transfer to another city . Both the concepts interest and incentive are closely related to motives . In psychology, several types of motives have been distinguished . Internal or intrinsic (also called genuine) motives are related to the activities themselves.20 for example, a phd student is motivated to create important insight and knowledge . This will often include hard work and struggle to reach the desired outcome that is intrinsically connected to the activity itself . In contrast to internal motives are the so - called instrumental motives, where the motive bears no intrinsic relation to the activity itself . Some instrumental motives could be that the phd student wants to get promoted, get a specialist position, or a good salary . Internal motivation predicts positive outcomes, while multiple motives may have negative impact in an educational context.20 idea: if the candidate has a good idea of what he or she wants to investigate, has sought out, and read a selection of the literature on the question to be researched, and if the idea is realistic, the probability of success is good . If, in addition, the idea is focused, the prognosis is better than if the idea is broad and diffuse . Initiative: if the student shows initiative, is always well prepared, drafts proposals, and meets deadlines, the probability of success is far more likely than for a reactive student who expects proposals to be provided by a supervisor who always has to take the initiative . The prognosis is, as expected, far better for proactive personalities than for reactive personalities . Personal characteristics predictive of success in a phd program are responsibility and realistic self - confidence . The prognosis is good for students with their own clearly grounded approaches to research, the research project, and the supervisor . The prognosis is also good for students who take responsibility for themselves and their programs and understand that obstacles and challenges help to drive personal development in the research process . The role of supervision should be very clearly defined as a professional relationship rather than a tutorial friendship.15 this commentary does not cover supervision, which is an independent discipline.15,21 clear communication is important in the relationship between supervisor and student . Expectations must match, and a framework must be agreed upon for the supervisory process . As stated earlier, it is important for a supervisor to have the right interest, competencies, and knowledge in relation to a given phd project . At the same time, the student s personal integrity is a prerequisite for the necessary discipline and time management (a major problem for many). Interpersonal relationships: the ability to communicate well and share knowledge plays a major role in successful interaction with a supervisor, other phd students, and employees in the department . During interviews, i try to uncover the ability to maintain and develop positive interpersonal relationships, relying on the following words as signals: motivation, willpower, sensitivity, expectations, psychological balance, loyalty, frequency and duration of previous positions, and earlier conflicts . If all the i - determinants discussed previously are present, the likelihood of success in a phd program is high . In my daily work, i operationalize my evaluation of applicants to a phd program by scoring them on each of the six i - determinants on a scale of 15, where 1 is lowest and 5 is best . Not surprisingly, a score> 20 is a good determinant of success in a program, while a score <18 is a strong predictor of less good outcome (predictive value 0.69). If the score is> 23, the predictive value is ~90%95% with the lowest value for incentive and highest for initiative . For a cut point of 23 points, the negative predictive values are between 0.26 and 0.53 . In a multiple logistic regression model, the estimates are imprecise but the strongest positive predictors are interpersonal relationships, interest, and incentive, while idea is a bit weaker predictor, but the discriminatory interpretation should be cautious . The candidate has completed a phd program, and it is known whether he or she completed the program on schedule, published papers in peer - reviewed journals, was awarded grants, and whether the process went smoothly . An overlooked and underprioritized area in international research is development of postdoctorals as research leaders . Of course, the i - determinants described previously for phd students also play a very important role in postdoctoral research . However, it is a considerable leap from being a phd student to being a postdoctoral . A well - developed capacity for critical thinking, conceptualization, and reflection is an important predictor for success in independent research . This must be combined with independent, innovative thinking and the ability and confidence to challenge existing hypotheses . At the postdoctoral stage, candidates must examine whether they can be research leaders, with the ability to supervise others and to integrate them into a research team . Understanding of leadership is essential and required, that is, as a process of inspiration, influence, setting a course, working with others, and thereby creating value . On this basis, i developed a series of i - determinants for postdoctorals (figure 3), identified through interaction with> 100 researchers at the postdoctoral level or higher with whom i have worked with over the years, nationally and internationally . Identity, independence, and image (self - image): postdoctorals who are big picture thinkers and aware of their own reputation, and their value in the research community, in a realistic way, often develop into successful independent researchers . Those who are committed to self - management and interested in leadership in research institutions possess essential positive characteristics . As well, postdoctorals who are persistent, who can tolerate refusals of papers and grants, and who can revise up to 2030 drafts of a paper before it is submitted, also possess qualities required for success as research leaders . As well, the successful researcher is highly inquisitive and consciously sets time aside each day to learn something new . A certain degree of isolation, loneliness, and opposition is part of a postdoctoral program, which must be tolerated and handled by a successful postdoctoral.implementation: ability to implement decisions and projects and see them through to completion is important determinants of a successful postdoctoral . In many cases, the ability to set priorities and manage time is essential determinants of the ability to complete one s research . Poor time management is a common problem, especially among phd students and postdoctorals, although they spend many hours on their work.innovative and important topics: if a postdoctoral published papers in leading peer - reviewed international journals as a phd student and is able to formulate projects building on this experience, or in new areas, he or she is likely to have the skills characterizing a successful research leader.in-depth knowledge of research topic: able and productive researchers are thoroughly familiar with their research field, including its textbooks and key publications.4,8 they have a clear, focused research agenda, possibly with multiple subagendas.8interactive and integrated with the scientific community: successful postdoctorals are well mentored and supported by their own department and university . They present their results at scientific meetings and may participate in the work of professional associations . They know they have already gained experience in providing supervision.internationally oriented: periods spent in another international research environment during a phd program are positive predictors of successful postdoctorals . A change in research environment, when the candidate has contributed personally to finding the necessary funding, is an experience that cannot be overvalued . Participation in collaborative international research is a key predictor of a postdoctoral s future success . Identity, independence, and image (self - image): postdoctorals who are big picture thinkers and aware of their own reputation, and their value in the research community, in a realistic way, often develop into successful independent researchers . Those who are committed to self - management and interested in leadership in research institutions possess essential positive characteristics . As well, postdoctorals who are persistent, who can tolerate refusals of papers and grants, and who can revise up to 2030 drafts of a paper before it is submitted, also possess qualities required for success as research leaders . As well, the successful researcher is highly inquisitive and consciously sets time aside each day to learn something new . A certain degree of isolation, loneliness, and opposition is part of a postdoctoral program, which must be tolerated and handled by a successful postdoctoral . Implementation: ability to implement decisions and projects and see them through to completion is important determinants of a successful postdoctoral . In many cases, a phd program will have clarified whether these qualities are present . The ability to set priorities and manage time is essential determinants of the ability to complete one s research . Poor time management is a common problem, especially among phd students and postdoctorals, although they spend many hours on their work . Innovative and important topics: if a postdoctoral published papers in leading peer - reviewed international journals as a phd student and is able to formulate projects building on this experience, or in new areas, he or she is likely to have the skills characterizing a successful research leader . In - depth knowledge of research topic: able and productive researchers are thoroughly familiar with their research field, including its textbooks and key publications.4,8 they have a clear, focused research agenda, possibly with multiple subagendas.8 interactive and integrated with the scientific community: successful postdoctorals are well mentored and supported by their own department and university . They present their results at scientific meetings and may participate in the work of professional associations . They know internationally oriented: periods spent in another international research environment during a phd program are positive predictors of successful postdoctorals . A change in research environment, when the candidate has contributed personally to finding the necessary funding, is an experience that cannot be overvalued . Participation in collaborative international research is a key predictor of a postdoctoral s future success . Regardless of the framework for research, the personal characteristics of a researcher play a very important role in the quality of the research . Our knowledge is limited about ways of selecting phd students and postdoctorals at the beginning of their research careers, who will produce the greatest value for society . The application of the principles discussed in this paper hopefully will allow decisions to be reached in a more evidence - based way.
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Distal radius fractures of the upper extremities are relatively common accounting for approximately 17% of all fractures in patients visiting the emergency room (1). In recent years, osteoporotic fractures have become a major health issue; and many studies have been done on relationship between distal radius fractures and osteoporosis . However, there have been still controversies whether this relationship is statistically significant or not (2,3,4). Also, the prevalence of low bone mineral density (bmd) and osteoporosis in patients with low - energy distal radial fractures compared with that of normal korean population without such fractures is not well known . In previous study, lee et al . (5) assessed age- and site - related bmd and the prevalence of osteoporosis in korean women with a distal radius fracture, and compared these with reference data derived from a study done in a large population - based cohort in korea (6). For the study, they recruited only small number of 54 consecutive korean women, 50 to 79 years of age, with a distal radius fracture . However, in this study we recruited large number of 206 consecutive korean women patients . This study has shown that bmd is lower in patients with a distal radius fracture in women younger than 60 years of age or over 70 years of age than in normal controls . In addition, low - energy distal radius fractures are predictive of future 15 years prior to osteoporotic hip and spine fractures (7,8,9). Considering these findings, it is important to analyze the relationship between bmd and distal radius fractures in postmenopausal women for the prevention of secondary fractures (10). The aim of this study was to compare the bmd and prevalence of osteoporosis of postmenopausal korean women with low - energy distal radius fractures with those of aged - matched normal korean women . We retrospectively reviewed 206 patients who visited our hospital between march 2006 and march 2010 because of distal radius fractures . Inclusion criteria of this study were low - energy distal radius fractures caused by out - stretched injury of the wrist and postmenopausal women older than 50 years of age . Patients previously diagnosed systemic diseases affecting bone metabolism (e.g., metabolic bone disease, renal osteodystrophy) or those who had been taken steroid for extended period were excluded from this study . We also excluded patients with previous distal radius fractures, those with high - energy injuries caused by a vehicle accident or fall, those with pathologic fractures, and those who had previously diagnosed osteoporosis . The bmd was measured at the spine and proximal femur using a lunar prodigy advance dual energy x - ray absorptiometry (ge healthcare, madison, wi, usa). At the spine, bmd values were obtained from the first to the fourth lumbar vertebral body . At the proximal femur, bmd values were obtained from the femoral neck, ward s triangle, and the intertrochanteric area separately . For each area, the t - score and z - score, which represent differences in bmd from the maximum bone density of young and same aged people, respectively, were calculated . According to the diagnostic criteria of the world health organization (who), osteoporosis was defined as any t - score lower than -2.5, osteopenia as any t - scores between -2.5 and -1.0, and normal bone density as any t - scores equal to or greater than -1.0 (11). Patients were divided into three groups by age; group 1 (50 - 59 years), group 2 (60 - 69 years), and group 3 (70 - 79 years). The bmd values and prevalence of osteoporosis in each age group were compared with those of age - matched control . The bmd values and prevalence of osteoporosis of the control were based on the reference data from healthy community dwelling korean population (6). These reference data were obtained from a study that used a large cohort of community dwelling korean women, 20 to 79 years of age . This study included the entire female age group, including women with osteoporosis, and had 599 women 50 to 59 years of age, 894 women 60 to 69 years of age, and 313 women 70 to 79 years of age, and it provided age- and site - specific bmd reference values . The equipment used in this study was dpx bravo (ge healthcare, madison, wi, usa), which is small and measures only hip and spine bmd . Results are reported as means with standard deviations or frequencies with 95% confidence intervals (ci). Continuous variables between patients and controls were compared with use of independent - sample t tests . Chicago, il, usa), and p - values <0.05 were considered statistically significant . This study protocol was reviewed and approved by the institutional review board of asan medical center (irb no . Informed consent was waived for this study since it is an observational study with minimal risk . Results are reported as means with standard deviations or frequencies with 95% confidence intervals (ci). Continuous variables between patients and controls were compared with use of independent - sample t tests . Chicago, il, usa), and p - values <0.05 were considered statistically significant . This study protocol was reviewed and approved by the institutional review board of asan medical center (irb no . Informed consent was waived for this study since it is an observational study with minimal risk . The mean height, weight, and body mass index (bmi) of the total 206 patients were 155.7 5.2 cm, 55.7 5.6 kg, and 23.1 2.8 kg / m, respectively . The differences of the mean height, weight, and bmi in each group were not statistically significant compared to control group (table 1). In group 1, except for the spine, all bmd values of the patients were significantly lower than those of age - matched control . While the bmd values in groups 2 and 3 were lower than those of age - matched controls, those differences were not statistically significant (table 2). L1-l4, from the first lumbar vertebrae to the fourth vertebrae; sd, standard deviation . One hundred and six (51.5%) of the 206 patients were newly diagnosed with osteoporosis . Osteoporosis was diagnosed most frequently at the ward s triangle (101 of 206 patients; 49.0%), followed by the spine (93 of 206 patients; 45.1%), the femoral neck (14 of 206 patients; 6.8%), and the trochanteric area (9 of 206 patients; 4.4%). Compared with age - matched controls, all groups had significantly higher prevalence rates of osteoporosis at the ward s triangle; however, the prevalence of osteoporosis at the spine, femoral neck and trochanteric area were not significantly different from those of age - matched controls (table 3). We analyzed the bmd and prevalence of osteoporosis in postmenopausal korean women with low - energy distal radius fractures . The patients were divided into three groups by age, and results were compared with age - matched normal korean women without fractures . This study showed that prevalence of osteoporosis at all measurement sites in the patients was not significantly lower than those of age - matched controls except at the ward s triangle . The ward s triangle, however, is not considered by international society for clinical densitometry to be a suitable site for bone density measurement, because the rate of osteoporotic prevalence at this site can be overestimated (12,13). In this regard, our results seems to be against the previous studies, which showed the prevalence of osteoporosis in patients with distal radius fractures to be higher than that of the control (3,14,15,16). In this study, however, 106 new cases of 206 patients (51.5%) of distal radius fractures were diagnosed as osteoporosis . The rates of newly diagnosed osteoporosis were 21 of the 71 patients (29.6%) aged 50 - 59 years, 51 of the 93 patients (54.8%) aged 60 - 69 years, and 39 of the 42 patients (92.8%) aged 70 - 79 years . Although the prevalence of osteoporosis of patients in our study was comparable with those of age - matched controls at all measured sites, except the ward s triangle, our results indicate that osteoporosis should be evaluated and managed by screening bmd in patients with distal radius fractures . Our study results showed that the bmd values were lower in all measured area in all patients groups; however, it was interesting that only the bmd in the femur area in group 1 was significantly lower than age - matched control . There has been still inconsistency in correlation between low bmd and age . In one study, patients younger than 65 years of age with distal radius fractures had significantly low bmd values, whereas low bmd values associate with older patients in another study (2,17). Our results are partly consistent with those of a previous study, which showed younger postmenopausal women with distal radius fractures to have low bmd values . The rate of bone loss in lumbar vertebrae decreases after menopause and stabilizes in women at 60 - 69 years of age, whereas in the femur it continues or accelerates with age (18,19). Therefore, postmenopausal women at 50 - 59 years of age with low - energy distal radius fractures should be evaluated for osteoporosis to prevent other osteoporotic fractures such as those of the hip and spine . The bmd of women peaks in their 30s and declines thereafter with age, greatly decreasing during the perimenopausal period (20). While healthy korean women have peak bmd values, bone loss is more rapid after 50 years of age compared to caucasian and lebanese women (6). Early detection of osteoporosis in younger postmenopausal women with distal radius fractures may help to prevent future osteoporotic fractures . Low bmd values associate with the severity of osteoporotic distal radius fractures (21). In contrast with distal radius fractures, hip and spine fractures were associated with an increased mortality (22). Our results indicate that an osteoporotic hip fracture can occur after a distal radius fracture in younger postmenopausal women with distal radius fractures . The major limitation of this study was that normative data were based on the reference data from korean community dwelling population (6). Bmd values obtained by different densitometers cannot be compared because technical differences in the devices exist . Nevertheless, we were able to compare bmd values with reference data because the densitometer and measurement sites were identical.
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With the rapid development of the image processing, reverse engineering, computer - aided design, and image guidance play an important role in dental implant surgery . A number of dental and jaw imaging modalities, including video imaging, computed tomography (ct), and magnetic radiotherapy imaging (mri), are used in image - aided dental implant surgery . Performing the multimodality image registration from different imaging devices has complementary significance for guiding dental implant surgery . When dentists design schemes, they want to consider the morphology of the soft tissue and the hard tissue together . Ct image and visible spectrum image fusion models can multiply various factors and help the dentists achieve the optimal implant scheme . Traditional registrations of cone beam computed tomography (cbct) and other three - dimensional scanner models mainly rely on the fiducial markers method, which need to manually select the feature points . In [68], manual feature selecting methods have been tried for the registration of the mandible ct and plaster cast of the dental models . Because the images are complex and the location accuracy is limited by the experience of the operator and the operating state, the manual feature selecting method might provide misleading information and lead to instability problems . Designed an occlusal form board, with titanium spheres markers, to match the image using the titanium spheres markers as feature points . In this model, the position of makers is stable, but the drawback is the extra burden for patients . The methods mentioned above cannot perfectly meet the needs of developing image - aided dental surgery . Two main difficult problems are taken out in the process of the registration; one is the feature point selection and the other is the merge of different modality images with different resolutions . Iterative closest point (icp), in recent years, many researchers have extended or improved the traditional icp algorithm . Proposed the fractional icp (ficp) algorithm to trim abnormal points effectively, which enhanced the robustness of the icp algorithm . Choi et al . Proposed the improved k - d trees traversal method to accelerate finding process of the nearest points . Bae and lichti further extended the improved icp method based on the boundary feature points of the point cloud, which improved the efficiency and accuracy of traditional icp algorithm greatly . The aim of this work is to develop an improved iterative closest point (icp) algorithm in terms of the problem of registration with different image resolutions . Firstly, extract feature point set based on curvature characteristics, and then achieve the registration results by two procedures: the coarse registration and the precise registration . To verify the reliability of the improved icp algorithm, we test it with 3d cbct dental model and ineos blue - light scanned model . These two models provide reconstructed ct dental data and visible light scanned dental - surface data, respectively . Experimental results show that the accuracy is increased significantly and this solution meets the requirements of the image - assisted surgery . Section 2 provides an overview of the icp algorithm and proposes an improved icp algorithm . Section 4 reports the results of experiments conducted on the cbct model and the ineos blue scanned model and provides the discussions . Generally, different medical imaging equipment differs in imaging mechanism, which contributes to different emphases on image information and makes image resolution difference . Thus, it results in complex difficulties in the image fusion; for example, the preprocessed 3d cbct dental model contains two parts: dental crowns and roots, while the 3d ineos blue - light dental model contains dental crowns only . Additionally, the resolution of the cbct model is 0.625 mm, while that of ineos blue - light scanner is 0.1 m . In view of data characteristics above first of all, feature point set of the three - dimensional surface images was extracted based on curvature characteristics to realize the coarse registration and to achieve the transformation matrix . Then, the feature point set and reference point set, obtained by the initial registered transformation, are processed in the precise registration step using icp . Icp algorithm is commonly used as registration method, which enables the minimum mean square error in the distance of corresponding point or point surface between two point sets approaching the minimal by iterative calculations . It repeats the process of calculating the optimal rigid transformation of the corresponding point set until convergence criterion is satisfied; that is, it has met the precision of registration . The transformation relation is shown as function (1), and the convergence criterion is shown as function (2): (1)qi = rpi+t,(2)e=i=1n||qi(rpi+t)||2, where pi and qi present point sets corresponding to two images, respectively, r is a rotation matrix, t is a translation vector, and e is the error . When the error reaches the minimum, the convergence criterion meets requirement . The traditional icp algorithm used in registration requires one of the two point sets to be the subset of the other one . The relatively initial positions of two point sets are limited strictly in icp, which ensure no big gap between the two point sets . When the constraints are unsatisfied or the huge deviation exists, it will affect the processing results and make the registration unreliable . The data of the cbct dental model and the ineos blue scanned model are both stored in stl file form and 3d models . The image surface discrete curvatures are sampled as the feature set . Because the stl model is comprised of many triangular meshes, an improved voronoi method is implemented to calculate vertex curvature . As shown in figure 1, v0 is a vertex of the network model . The curvature of v0 can be calculated by (3)k=2jja (j=1,2,,n), where j is the angle between the side v0vj and the side v0vj+1, and a is the area of all triangle meshes which are connected with v0 . Set as the threshold . When the curvature of one point is greater than, it will be marked as feature point and put it into alternative feature list . The feature point set is defined as p = {pi | pi r; i = 1,2, 3,, np}. The improved three point's translational transformation method is used in this paper based on curvature characteristics to calculate rotation matrix r and translation vector t, so as to realize the semiautomatic extraction of corresponding points for two dental image models . At the same time, this method can reduce the interference of human factors in the matching process and enhance the accuracy of coarse registration . For the two image models, select three points, whose curvatures in the top 10 percent and distributions are homogeneous, as the reference points from the reference model and the corresponding points are in target model . After achieving reference points, we calculate the curvature of each vertex in target model and compare it with their value of the reference points . When the error is smaller than the setting threshold, mark this point as the centralized point of corresponding points and put it into corresponding points list . Due to the fact that there are some feature points with the same or similar curvature, it is common to lead to one reference point corresponding to some points . So we construct more powerful constraint condition, the distance of three points, and the area of triangle of three points, further to select the corresponding point set . This constraint condition is independent of rigid transformation and will improve the reliability and efficiency of determining corresponding points, and its specific steps are as follows . Assume there are three known reference points m1(x1, y1, z1), m2(x2, y2, z2), and m3(x3, y3, z3) and three corresponding points n1, n2, and n3 that need to be determined . Let a, b, and c represent the distance between m1, m2 or m1, m3 and m2, and m3, respectively . The computing function of the distance and the area are shown as follows: (4)a=(x1x2)2+(y1y2)2+(z1z2)2,b=(x1x3)2+(y1y3)2+(z1z3)2,c=(x2x3)2+(y2y3)2+(z2z3)2,s=+b+c2(+b+c2b)(+b+c2c). The distance constraint condition is: (5)|dmimjdninj|<, (i, j=1,2,3), where dmimj and dninj are the distances between any two points of the reference points m1, m2, and m3, and the corresponding points n1, n2, n3, respectively . The area constraint condition is shown by: (6)|s(m)s(n)|<, where s(m) is the area of triangle composed of m1, m2, and m3; s(n) is the area of triangle composed of the three corresponding points n1, n2, and n3; and is the set error . Through the above stated constraint conditions, we can select the corresponding points n1, n2, and n3 of the reference points m1, m2, and m3 . Local coordinate system of one of the two image models is built by reference points in the reference model . Assume that m1 is the origin of coordinate and the direction from m2 to m3 is x axis direction . Local coordinates can be expressed by tm = (t1(m), t2(m), t3(m)), where (7)t1(m)=m3m1|m3m1|,t2(m)=(m3m1)(m2m1)|(m3m1)(m2m1)|,t3(m)=t1(m)t2(m). Similarly, the local coordinate built by corresponding points n1, n2, and n3 is tn = (t1(n), t2(n), t3(n)). By formula deducing, we could obtain rotation matrix r and translation vector t corresponding to the transformation of coordinate tm to tn: (8)r = tn(tmt),(9)t = n1+n2+n33r(m1+m2+m3)3 . Then, the matrices r and t are applied to the two target point sets and we could realize the course registration . The accuracy of coarse registration cannot satisfy the ultimate requirements [16, 17], so icp algorithm is adopted for getting higher registration accuracy . Assume that the feature point sets of blue - light scanner dental model and cbct dental model are p = {pi | pi r; i = 1,2, 3,, np} and q = {qj | qj r; j = 1,2, 3,, nq}, respectively, where np and nq stand for the number of the elements (np nq). R, t are the rotation matrix and translation vector, which are obtained through the coarse registration . Then carry out the icp algorithm to make precise registration that consists of the following stages . Step 1given that k = 0, p = p, p = {pi | pi r; i = 1,2, 3,, np}, r = r, and t = t, use k - d tree method to calculate the corresponding point set d = {qj | j = i = 1,2, 3,, np}, which belongs to q, and each point qj is with the least euclidean distance to the point pi (i = 1,2, 3,, np). Define the initial residual error; that is, r = (1/np)i=1||pi d(i)|| . Given that k = 0, p = p, p = {pi | pi r; i = 1,2, 3,, np}, r = r, and t = t, use k - d tree method to calculate the corresponding point set d = {qj | j = i = 1,2, 3,, np}, which belongs to q, and each point qj is with the least euclidean distance to the point pi (i = 1,2, 3, define the initial residual error; that is, r = (1/np)i=1||pi d(i)|| . Step 2use r and t transform point set p to obtain the point set p, where p = rp + t. then calculate the corresponding point set d = {qj | j = i = 1,2, 3,, np}, which also belongs to q, and each point qj is with the least euclidean distance to the point pi . Then compute the residual error r = (1/np)i=1||pi d(i)|| . Use r and t transform point set p to obtain the point set p, where p = rp + t. then calculate the corresponding point set d = {qj | j = i = 1,2, 3,, np}, which also belongs to q, and each point qj is with the least euclidean distance to the point pi . Then compute the residual error r = (1/np)i=1||pi d(i)|| . Step 3judge whether the precision meets the requirements after iterating k(k 0) times . R |, is satisfied stop iterating . Otherwise, continue to step 4 . Step 4calculate the rotation matrix r and translation vector t of transform relation between p and d through the singular value decomposition (svd) method . Then renew k = k + 1, and transfer to step 2 . Calculate the rotation matrix r and translation vector t of transform relation between p and d through the singular value decomposition (svd) method . Then renew k = k + 1, and transfer to step 2 . The singular value decomposition (svd) method, used to calculate the rotation matrix and translation vector, is demonstrated in the appendix . The experimental procedures include acquiring data, preprocessing, extracting feature points, and registration . Firstly, using 3d sirona ineos blue blu - ray scanner (resolution is 0.02 mm) patient's plaster dental model is scanned . Next, the own software of scanner is applied to obtain stl format dental file . Geomagic software is used to preprocess the exported file, and then, export the preprocessed file, which is stl that format includes dental crown only . Sirona galileos system (resolution is 0.1 mm) is adopted to scan the same patient's mandible to obtain the ct scanning data . The data is dicom format and then it is imported into commercial processing software called mimics, which is used to reconstruct 3d ct model . The ineos blue dental model, showed in figure 4 as the sample, set the threshold = 0.01 . Calculate the vertex curvature of the dental crown using the improved voronoi method mentioned before . When the curvature value of any point is more than, mark it as a feature point and put it into point set list, which is denoted by p. the red dots showed in figure 6 represent the feature points . The resolution of 3d ineos blue dental data is higher than cbct data, while cbct model includes the integral dental data . The improved three point's translational transformation method is used to realize coarse registration, and the results are shown in figure 7 . Select three corresponding points m1, m2, and m3 as reference points from cbct dental model and set the threshold 0.00015 . Then extract the corresponding points n1, n2, and n3 and calculate rotation matrix r and translation vector t. the results are listed in table 1 . Formula (8) is used to realize coarse registration, and figure 8 is the result of coarse registration . Based on the results of coarse registration, the precise registration is carried out for improving the accuracy of registration further . Figure 9 is the result, which reveals that the two models can be fused more accurately . Supported by the vc6.0 platform, we used vc++ and opengl to conduct simulation and experiments . We show dental models registration results to evaluate the accuracy of our algorithm, and the average distance of corresponding points is calculated . For the first experiment the average distance of the coarse registration, shown in table 2, is 0.7291 mm while it is reduced to 0.3835 mm after precise registration . Other four groups of patient's dental image data are carried out by the same procedure, which also conclude ineos blue scanner and cbct 3d dental models and yield the same conclusion . The total average distance of the precise registration is 0.8266 mm, compared with 1.4486 mm of the coarse registration . Comparing with the coarse registration accuracy, the precise one is improved significantly, which basically meets the requirements of helping dentist for designing remedies . Considering the characteristic of the stl model data itself, we take the cbct 3d dental model as the reference model and adopt the constraint - based curvature method . As mentioned earlier, extract the corresponding feature points automatically in the ineos blue scanner dental model . Experimental results show that the method could extract the corresponding feature points correctly and be operated simply and reliably . Moreover, it efficiently overcomes the shortages of both the traditional surface - based marking method and the corresponding manually selected feature points method . Multimodality image registration and fusion technology can make full use of the complementary characteristics which are provided by different imaging models . The technology could perfectly integrate the image data to provide abundant information for clinical diagnosis . The improved icp method proposed in this paper realized the feature points extraction and image registration successfully between the ineos blue scanner dental model and the cbct 3d dental model . Besides, it provides reliable registration method in the computer - aided dental implant design system . The research and the method in this paper will be the reference and application for similar studies and other multimodality image registrations . Imperfectly, in the process of extracting feature points with curvature characteristics, the threshold has to be appropriately adjusted according to the model complexity and the accuracy requirements, which make the registration accuracy to a larger degree depend on the reasonable choice . As the traditional icp algorithm cannot realize the image registration with different resolution, we propose an improved icp algorithm in this paper . The procedure of this algorithm used in the registration of 3d cbct dental model and 3d ineos blue scanning dental model is discussed in detail.
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Recurrent disc herniation has been reported in 5 - 15% of patients after disc excision, which represents the most common cause of unsatisfactory results1,2,3,5,7,9,10,11,12,15). The recurrence of symptoms after an initial period of symptomatic relief can be caused by a recurrent disc herniation, new disc herniation at a different disc level, epidural fibrosis, arachnoiditis, foraminal stenosis, or segmental instability9,14). Several conditions have been termed as " recurrent disc herniation " including recurrence at the same disc level and on the ipsilateral side as the primary herniation, contralateral herniation at the same disc, or a new herniation at a different level3). The clinically appropriate definition of " recurrent disc herniation"is that a disc herniation at the same disc level with an ipsilateral side as a previously operated on . There have been numerous studies of recurrent disc herniation, but these have analyzed mixed patient populations and included patients with different causes of failed back surgery, as well as those with herniation at a different level or contralateral side compared with the primary discectomy . We designed this study to analyze the factors influencing the clinical outcome and efficiency of repeat discectomy for recurrent lumbar disc herniation at the same level and on the ipsilateral side after primary discectomy . From january 1990 to september 2011, a series of 2816 patients underwent lumbar discectomy for disc herniation . Of these patients, 52 patients underwent on repeat discectomy due to recurrent disc herniation . Recurrent lumbar disc herniation was defined as disc herniation at the same level and on the ipsilateral side . The symptomatic relief period after the patients with a disc herniation occurring at a new level or contralateral side with respect to the primary discectomy were excluded from this study . Clinical and radiological factors associated with recurrent disc herniation were evaluated using preoperative clinical medical charts and radiological examinations . The evaluated demographic and clinical parameters were as follows: age, sex, smoking habit, diabetes mellitus (dm), traumatic event, symptom duration before repeat discectomy, and time to repeat discectomy from initial discectomy . The extent of disc herniation was evaluated and classified as protrusion, extrusion or sequestration . A disc was considered protruded if the greatest plane, in any direction between the edges of the disc material beyond the disc, was less than the distance between the edges of the base when measured in the same plane . A disc was considered extruded if, in at least in one plane, any one distance between the edges of the disc material beyond the disc space was greater than the distance between edges of the base measured in the same plane . A sequestrated disc was one in which the disc material was clearly separate from the originating disc2). The pain was evaluated by a 10-point visual analogue scale (vas) scoring (0 - 10) before and after repeat discectomy, and differences of vas score were measured (vas). Postoperative satisfaction was evaluated using macnab criteria (excellent, good, fair and poor). Clinical success was defined as if patients showed (1) more than 4 point improvement in vas scores, (2) excellent or good in macnab criteria and (3) no major complication related to the surgery . Statistical analysis was done using fisher's exact test or the cochran - mantel - haenszel test for categorical variables, anda t - test for continuous variables . The group consisted of 35 men and 17 women and their average age at repeat discectomy was 40.4 years (range 18 - 72 years) (table 1). The level of recurrent disc herniation was l4/5 in 31 cases, l5/s1 in 17 cases, l3/4 in two cases . The mechanism of onset was a car accident in 4, a sporting accident in 2, and a fall in 2 . The incidence of chronic smoking was 8 (15.4%), and of diabetes mellitus 2 (3.8%). The time from prior surgery to the repeat discectomy averaged 24 months (table 1). The outcome of postoperative clinical symptoms was evaluated using macnab criteria, and the results were as follows: excellent (28), good (22), fair (1), and poor (n=1). The patient with grade poor had a post - operative infection, and required revisional surgery . The patient with grade fair required further pain relief such as pain block . The mean preoperative vas score in revision surgery was 6.98, and this improved after the revision surgery to 2.1 . Forty - seven patients out of 52 showed improvement of more than 4-point on vas scale . No factors affected clinical outcome of repeat discectomy for recurrent disc herniation assessed by macnab criteria and vas score according by age, sex, diabetes mellitus, smoking, time interval between repeat and primary discectomy, symptom durationbefore the repeat discectomy, and extent of disc herniation . Traumatic event showed significantly affected the vas score, however not significantly on the macnab criteria . According to clinical success, traumatic event was the only statistically significant factor that showed poor clinical outcome after repeat discectomy (p=0.022) (table 3, 4, 5, 6). Conventional open lumbar discectomy has been used as a surgical treatment for a herniated lumbar disc since the initial ofthis discovery disease entity more than 70 years ago10). However, the revision rate has ranged between 2 - 19% in previous studies4,5,6,7,8,9). There are numerous operative methods that can be done on patients with recurrent lumbar disc herniation such as repeat discectomy, percutaneous endoscopic discectomy, posterior lumbar interbody fusion and transforaminal interbody fusion . The current study assessed patients with proven recurrent disc herniation at the same location as a previous surgically treated herniation . Therefore, the authors were able to study the clinical outcomes after revision surgery for a true recurrent disc herniation . It has been shown that the outcomes of patients surgically treated for recurrent disc herniation vary widely . Patients with recurrent herniation are often reviewed together with those presenting with herniation occurring at a new level or with those in whom lumbar surgery has failed for another reason . Cinotti et al.3) prospectively analyzed patients with recurrent disc herniation at the same level as at primary discectomy, either on the same or opposite site, and found no significant difference in clinical outcome between patients with recurrentherniation and those without recurrent herniation . In another study, suk et al.14) showed that satisfactory results were obtained when conventional open discectomy was used for revision surgery for recurrent lumbar disc herniation, and that these were comparable to those obtained with primary discectomy . Erbayraktar et al.4) reported the findings of repeat discectomy in 22 patients with prior lumbar discectomy at the same or a different level, either on the same or on the opposite site . There were 28 excellent, 22 good, 1 fair and 1 poor macnab criteria . Clinical success was seen in 47 of the 52 patients (90%). In patients with diabetes mellitus, hospitalization is prolonged and there is a higher risk of postoperative infection as well as poorer results15). Simpson et al.13) reported that excellent and good outcomes in 39% of their patients with diabetes, while 95% of those without diabetes, after an initial discectomy . The patients with diabetes has poor outcome caused by diabeticpolyneuropathy before neural compression caused by disc disease . However, in our study, diabetes was not a statistically significant factor affecting clinical outcome . Predisposing factors associated with an increased risk of primary disc herniation are a constitutional weakness of the annular tissue, exposure to repetitive lifting, vibrations and smoking . Isolated trauma or injury seems to play a prominent role inthe etiology of disc herniation since this has been reported by 0.2 - 10.7% of adults with documented herniation3). We found that 15% of patients with recurrent herniation related the onset of radicular pain to traumatic event . This suggests that the annular incision performed at surgery makes the disc to be more susceptible to sudden prolapse, particularly under conditions of mechanical overload such as those occur during sports or lifting . In the present study, traumatic events were statistically significant factor affecting vas score, but not a statistically significant factor affecting macnab criteria . According to clinical success criteria in this study, traumatic events are the statistically significant factor for poor clinical outcome (p<0.05). That was vas score puts emphasis on subjective pain, combined musculoskeletal pain due to trauma . In macnab criteria, good was thatpatients have non - neuropathic pain, but postoperative pain was relieved . Thus, although pain due to disc disease was relieved, vas score not decreased . Morgan - hough et al.12) noted that patients with protrusion are almost three times as likely to require revision surgery asthose with extrusions or sequestrations . This finding could be because a protrusion represents the beginning of a process of serial fragmentation of disc material, whereas extrusion and sequestration occur at the end stages of this process . However, in our study, the extent of disc herniation was not a statistically significant factor affecting clinical outcome . There are many reports that analyzed the outcomes of repeat surgery in relation to the time interval between the primary and second operation . Some reported that shorter period of time (less than one year) between primary and second operation results the better clinical outcomes4,8). However, fandifio et al.5) reported that the clinical outcomes were independent of the time interval between the primary and repeat surgery, which showed the same result in this current study . Different factors such as patient's age, sex, smoking, traumatic events, operation time interval, symptom duration before the repeat discectomy that may influence the outcome of repeat discectomy for recurrent disc herniation have beenanalyzed in numerous studies, but still remain controversial . Some reports found no significant factors that influence the clinical outcomes after the repeat lumbar discectomy11,14). Some aspects of the current study design might have affected the validity and thus the results . They might have undergone primary surgeries for different indication, and different procedures might have been used . In addition, no factors were found to be statistically related to the outcome of repeated discectomy for recurrent disc herniation (p>0.05) except for traumatic events . The strength of this finding might be weak; that is the insignificant results of the other factors might have been due to an insufficient numbers of patients for detecting significant differences . More research was needed on the dural tear caused by epidural adhesion that important role in evaluating outcome of revision surgery . Conventional open lumbar discectomy performed as a revision surgery for recurrent herniation showed satisfactory results . Based on the results of this study, repeat discectomy can be recommended for the management of recurrent lumbar disc herniation.however, patients with traumatic event before revision seem to show poor clinical outcome . Further study is required to identify factors associated with the outcome of revision surgery for recurrent disc herniation.
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The first non - invasive treatment for urinary stones, developed in 1980 was extracorporeal shock wave lithotripsy (eswl). To this day, eswl remains the least invasive treatment for urinary stones and is considered the preferred treatment for the majority of urinary stones, especially those of the kidneys . More than 90% of urinary stones in adults might be suitable for eswl, but the number of eswl procedures is lowering in the era of flexible ureterorenoscopy and percutaneous nephrolithotomy . The introduction of affordable multifunctional lithotripters has made the eswl available to increasingly more urology departments worldwide . However, pain control during the eswl procedure remains a problem . Several anesthesia techniques have been used to provide sedation and analgesia . Some of the analgesic drugs administered for the eswl, however, carry the risk of respiratory depression, delayed discharge, and/or require unplanned hospital admission . Since 2013, pethidine in combination with diazepam and diclofenac sodium in combination with hyoscine - n - butyl bromide combinations have been standard analgesia for patients who underwent eswl in our department . Due to this reason the main purpose of this study was to compare two treatment modalities for pain control during the eswl procedure . This study was designed as a non - randomized prospective study and approved by the local ethics committee of our institution and performed in accordance with the helsinki declaration of the world medical association . Informed consent was obtained from all patients prior to eswl procedure . An electrohydraulic device (spark gap technology, elmed lithotripsy systems, ankara, turkey) was used . Between 2013 and 2014, a total of 220 patients received eswl for kidney stones that were between 42 and 200 mm . Stone burden was calculated by multiplying the longest horizontal and vertical axis of the stone viewed on plain film . A maximum of 3000 shocks were applied at 80 shocks per minute during each session or until complete disintegration of the stones . We measured oxygen saturation by pulse probe (pulse oximeter) that can be applied to the thumb of the patients . The main reason for using the pulse oximeter was to observe the changes in oxygen saturations which can be the result of respiratory depression, one of the side effects of pethidine . The pain control provided during the procedure included two different methods: diclofenac sodium in combination with hyoscine - n - butyl bromide was administered to the first group and pethidine in combination with diazepam was administered to the second group . One hour before the procedure, diclofenac sodium (75 mg / kg) was administered i m, and hyoscine - n - butyl bromide (20 mg / kg) was administered by iv infusion during the procedure in the first group . In the second group, one hour before the procedure, pethidine (1 mg / kg) was administered i m, and diazepam (10 mg) infusion was provided during the procedure . During the procedure, the side effects of the administered drugs including nausea, vomiting, allergic reactions, and dizziness were recorded by an eswl nurse who was educated beforehand about the side effects of the drugs in our study . The pain level of the patients at the end of the eswl procedure was determined using two different scales (in order to increase the accuracy of pain level): the wong - baker and the visual analogue scales (vas). The wong - baker scale is used for pain assessment with six different face forms that are scored on a scale from 0 (no pain) to 5 (worst possible pain) (figure 1). The vas is used for pain assessment with a visual image of a straight line 10 cm in length that is scored from 0 (no pain) to 10 (worst possible pain). The patient was asked to mark the pain level at a point on the straight line (figure 2). Patient comfort was determined by asking the following question: would the patient repeat the procedure without any changes?. The meaning of the observer answered the question did the patient finish the procedure?. If the patient tolerated the pain until the end of the procedure, then the possible answer was yes, and if not, no, not tolerated. At the end of three sessions, all patients were evaluated for the stone fragmentation rate, by plain abdominal x - ray, and the findings were recorded and analyzed . From the beginning of the procedure, all patients were followed - up by a nurse for pulse and blood pressure monitoring and presence of side effects such as: nausea, vomiting, dizziness, and respiratory depression . Statistical analyses of the means of continuous variables were performed with the student's t - test and mann - whitney test . Bivariate, multivariate, regression model and the pearson correlation tests were used for correlation among variables . A probability level of p statistical analyses of the means of continuous variables were performed with the student's t - test and mann - whitney test . Bivariate, multivariate, regression model and the pearson correlation tests were used for correlation among variables . A probability level of p there were 91 patients in the first group (diclofenac sodium + hyoscine - n - butyl bromide) (male / female: 63/28) and 129 (male / female: 83/46) patients in the second group (pethidine hcl + diazepam). The mean age with the sd according to the groups was 42.03 (16.43) and 42.56 (14.23), respectively (p = 0.8) the demographic characteristics of two groups are summarized in table 1a . The demographic characteristics of the patients as mean values with standard deviations the findings of the study with standard deviations there were no statistical differences between the groups with regard to values of oxygen saturation and pulse before the eswl procedure . However, the values at the end of the procedure were lower in the second group . The difference between groups with regard to oxygen saturation was not significant (p = 0.06); however, the difference in pulse rate was significant (p <0.001). The responses to the two questions, as noted previously, were higher in the second group (p <0.001) (table 1b). With regard to the pain scores, there was no statistical difference between groups in terms of stone size (p = 0.94, 115.88 32.156 mm 116.26 39.91 mm for group 1 and 2, respectively). The fragmentation rates during the first sessions were higher in the second group (p = 0.01) (table 2). There was no significant difference with regard to vomiting; however, the nausea rates were higher in the first group (p = 0.02) (table 1b). The pain scale scores showed no significant differences with regard to gender (wong - baker scale p = 0.06, vas scale p = 0.3) (table 3). There was no correlation between the bmi and pain scale scores according to groups (pearson correlation value: 1, p = 0.93). In addition, there was no correlation between stone size and the pain scale scores (pearson correlation value:0.03, p = 0.6). There was a positive correlation between age and both of the pain scale scores (pearson correlation value: 0.148, p = 0.02 for scale 1, and the pearson correlation value:0.13, p = 0.04, for scale 2). We found that pain control methods had an effect on vas score and fragmentation rate of the stones with multivariate analysis (p = 0.000 and p = 0.012, respectively and adjusted bmi had no effect on vas score and fragmentation rate (p = 0.727 and p = 0.166, respectively) (table 2). The results of multivariate analysis the pain scores according to gender (mean values with sd) there was no significant dizziness, respiratory depression or hypotension noted in any of the patients . The noninvasive eswl has become the procedure of choice for treatment of urinary system stones . The development of second and third generation lithotripters has eliminated the limitations associated with the dornier hm-3 (water bath, anesthesia). Simultaneously, the new development of endoscopic approaches (flexible ureteroscopes, holmium laser) has made endoscopic stone procedures less traumatic and an attractive choice because of the higher primary success rate . Most patients with urinary stones in turkey and developed countries prefer the eswl as first line management due to the complication potential associated with anesthesia [6, 7]. Eswl stone - free (sf) rates, for stones less than 2 cm, vary widely from 55% to 90% . Many factors influence eswl success rates, including the type of lithotripter used, patient characteristics such as bmi and age, as well as stone characteristics, such as size, location and hardness, any anatomic abnormalities related to the kidneys, and patient comfort during the procedure . Also, pettenati et al ., showed that the presence of a double j stent affected the efficacy of eswl in the treatment of lumbar ureteral stones . According to their study, logistic regression analysis revealed a higher failure rate when a double j stent was associated with a stone> 8 mm . Chaussy and thuroff showed that the need for analgesia during the eswl depends on the lithotripter used, the stone location, age, gender, and the number of shock waves performed . According to the results of this study, the pain scores were higher in women; however, this difference was not statistically significant (p = 0.06 for scale 1 and p = 0.3 for scale 2) (table 3). There was no correlation between the two groups with regard to bmi and stone size . However, there was a positive correlation between age and the pain scale scores in this study (pearson correlation value: 0.148 . Younger patients tolerated the procedures better than older ones; but this correlation was found to be weak . Thus, the preference for anesthesia during eswl has progressively shifted from general anesthesia to sedation, except for special situations depending on the patient . Combinations of a sedative hypnotic and opioid analgesic are frequently used to provide patient comfort . According to the european association of urology guidelines for urolithiasis, suitable analgesia is recommended because of its effect on treatment results by limiting pain - induced movements and excessive respiratory excursions as well as improving patients comfort . It is well known that acute pain results in shortness of breath and an increase in ventilation . According to the report of borgbjerg et al ., the kidney can mobilize about 2 - 4 cm depending on the depth of respiration . This finding can be associated with the degree of the perception of pain during the eswl . For example, in situations without good pain control, the number of shock waves focused on the stone decrease resulting in a lower fragmentation rate during the first session . The results of this study showed that the fragmentation rate in the second group (pethidine + diazepam) was higher than the first group (diclofenac + hyoscine - n - butyl bromide) (p = 0.01) (table 2). The use of diazepam likely reduced the depth of respiration by decreasing the patient's anxiety and pain . Therefore, reduced perception of pain during the eswl is essential for targeting and optimal fragmentation of stones during the eswl . According to the eau guidelines on urolithiasis, careful control of pain during treatment diazepam used in the second group reduced patient movement during the eswl procedure by providing the patient with sedation and relaxation . Various analgesic agents including opioids (morphine, pethidine and fentanyl), nonsteroidal anti - inflammatory drugs (nsaids - diclofenac, propofol, ketorolac, and piroxicam), local anesthetic agents and a number of combinations have been used during the eswl by various techniques such as general anesthesia, subcutaneous and intravenous injections, patient - controlled analgesia, and monitored anesthesia care . Cutaneous creams such as a eutectic mixture of local anesthesia (emla) whether used alone or in combination with oral nsaids have also been used and can reduce analgesic requirements . Topical application of a combination of dimethyl sulfoxide and lidocaine has also been found to be effective . According to the results of these studies, pain - control has been provided . While choosing the most suitable analgesic, one of the most important issues is to decrease patient anxiety and provide comfort so that repetitive sessions of eswl can be provided in cases where residual stone fragments need additional sessions . One of the reasons for adding a benzodiazepine, in this study, was to decrease the anxiety and perception of pain among patients . Mazdak et al . Used pethidine for pain - control during the eswl and the pain scores of their patients was 4.11 1.69 on the vas . The results of this study showed that the score could be reduced about two points, with the same pain scale, by combining pethidine and diazepam . According to the results of this study, the rate of additional sessions because of residual fragments was higher in the second group when compared to the first group; the difference between groups was significant statistically (p <0.001) (table 2). This result may be associated with the good pain - control in the second group during eswl . Although opioids provide effective analgesia, they are associated with significant complications such as respiratory depression, bradycardia, hypotension, nausea, vomiting, and an extended monitoring time . According to the results of this study, the nausea rates were 47.3% and 32.6% for groups 1 and 2, respectively (p = 0.02) (table 1b). This was associated with the colic pain that occurred during the eswl; likely due to mobilization of the stone fragments in the kidney . The sedation provided by diazepam in the second group reduced the nausea rates compared to the first group; however, there was no difference in vomiting between the two study groups . There were no differences between groups before the procedure, but after the procedure, although there was no difference in the blood oxygen saturation, the pulse rate was lower in the second group compared to the first group (p <0.001) (table 1b). We did not obtain a cut - off value on pain level with our data and we could not analyse the effect of double j stents on the treatment of stones with eswl due to the sizes of the kidney stones in our study (less than 2 cm). As is the routine for our department, we do not prefer to insert double j stents prior to the eswl in patients having stones less than 2 cm . We think that if they were included in the study, the results would be more significant . We did not obtain a cut - off value on pain level with our data and we could not analyse the effect of double j stents on the treatment of stones with eswl due to the sizes of the kidney stones in our study (less than 2 cm). As is the routine for our department, we do not prefer to insert double j stents prior to the eswl in patients having stones less than 2 cm . We think that if they were included in the study, the results would be more significant . The results of this study showed that a pethidine combination with diazepam was superior to diclofenac in combination with hyoscine - n - butyl bromide . By providing good analgesia and sedation,, the fragmentation rates and need for additional sessions to deal with fragments or hard stones were increased . Many types of analgesia have been applied by various techniques by urologists, excluding general anesthesia and deep sedation, however, there are no guidelines for pain - control . Additional studies are needed to determine the safest and most effective analgesia with regard to side effects, discharge time, easy usage (for urologists), and cost in patients undergoing the eswl procedure . The ideal analgesic that would offer optimal pain control, minimal side effects, and cost - effectiveness remains to be determined . Opioids administered using various techniques, provide effective analgesia, but they require active monitoring of the patient and have potential adverse effects.
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An esthetically pleasing smile requires gingival health, a proportional amount of gingival display during forced smiling and harmony among the size, shape, position, and color of the teeth . Excessive gingival display during smiling has been a case of esthetic embarrassment for many patients . Gummy smile is seen due to improper relation between the gingival tissue and the tooth, with gingival tissue in excess and tooth portion in a small amount . At least 50% of the patients exhibits some form of gingival display in a normal smile . However, exaggerated or forced smile patterns display gingiva in up to 76% of all patients . In absolute numbers, a normal gingival display between the inferior border of the upper lip and the gingival margin of the maxillary central incisors during a normal smile is 12 mm . In contrast, an excessive gingiva - to - lip distance of 4 mm or more is classified as unattractive by lay people and general dentists . A subset of this patient population exhibits an upper lip that lies far enough above the maxillary gingival zenith that it is described as unattractive . Gummy smile may not present with any pathologic difficulties, but definitely affects the patient's psychosocial behavior . It is the gingiva which dominates the visual feature in gummy smile when compared with teeth and lips giving an unaesthetic appearance . Patients having excessive gingival display because of delayed eruption of teeth can be treated by crown lengthening and gingivectomy, but some patients might require further treatment . A careful diagnosis and a proper treatment plan thus are very essential correcting the deformity . Diagnoses such as hypermobile upper lip, short upper lip, and/or vertical maxillary excess require surgical corrections . Moderate gingival display that is not skeletal in origin and vertical maxillary excess that ranges between 4 and 8 mm can effectively be treated by surgical repositioning of upper lip limiting the retraction muscles such as zygomaticus minor, orbicularis oris, levator anguli oris and levator labi oris . Conventional lip repositioning has been described in many case reports recently for the designing the gummy smile; however, only few have described the modified lip repositioning procedure . This case report describes the surgical technique and outcome of modified lip repositioning in the treatment of excessive gingival display . A 23-year - old male patient reported to the department of periodontology and implantology, vyas dental college and hospital, jodhpur (india), with the chief complaint of excessive display of gums while smiling . There were no significant medical or family history and the patient presented with no medical conditions that could contradict the surgical procedure . On extra oral examination, however, the high lip line was noted during smiling, which presented with a moderate gingival display . Intraorally, a moderate gingival display was seen during smiling, which extended from maxillary right first molar to maxillary left first molar . Also, there was slight higher pull of the upper lip on the right side near first molar when compared to the left side which gave an unesthetic appearance [figure 1]. Measurements such as gingival display, while forced smiling [figure 2] and the upper vermillion lip length were recorded with unc calibrated probe . There was a 6 mm of gingival display on forced smiling and 7 mm of upper vermillion lip length . The preoperative figure showing the gummy smile 7 mm of gingival display as measured by unc-15 probe informed consent was obtained after discussion of the benefits, possible complications, and alternatives to lip repositioning . Lip repositioning is a surgical way to correct gummy smile by limiting the retraction of the elevator smile muscles (e.g., zygomaticus minor, levator anguli oris, orbicularis oris, and levator labi superioris) and yet preserve the labial frenum . Local anesthesia (xylocaine 2% with epinephrine 1:80,000) was administered at the vestibular mucosa and lip from the maxillary right to left first molar . With the help of a sharp probe, bleeding points were induced at the mucogingival junction, which guided the first incision to be carried out . A partial thickness incision was made at the mucogingival junction from the mesial line angle of the right central incisor to the mesial line angle of the right first molar [figure 3]. A second partial thickness incision that ran parallel to the first incision and 1012 mm apical of the mucogingival junction was made in the labial mucosa . The incisions were connected at the central incisor region without involving the maxillary labial frenum and at the right first molar region creating a quadrilateral outline [figure 4]. The epithelium was then carefully dissected within this outline [figure 5], leaving the underlying connective tissue exposed [figure 6]. The same procedure was carried out on the left side at the mucogingival junction by making an incision from mesial outline of left central incisor to the mesial outline of left first molar . The next partial thickness incision was the one parallel to the first incision and 1012 mm apical to the mucogingival junction . The second strip of epithelium was then removed by careful dissection [figure 7]. The first partial thickness incision on mucogingival junction from mesial line angle of right central incisor to mesial line angle of right first molar the second partial thickness about 810 mm above the first incision and two vertical incisions to join at both the ends the strip of the epithelium which was removed the partial thickness dissection which was carried out the same procedure was done on the left side leaving the intact frenum the parallel incision lines were approximated with interrupted stabilization sutures (silk 4/0) [figure 8]. Patient was discharged with all postsurgical instructions and medications for 5 days which included analgesic (ketorolac trimethamine 10 mg twice daily for 3 days), antibiotic (amoxicillin 500 mg tds for 5 days), along with cold packs extra orally to decrease postsurgical swelling . The 4 - 0 mersilk sutures were placed patient was recalled after 1-week for a follow - up . The patient reported with mild pain and tension at the surgical site during the 1 week after surgery . It was seen later that the suture area healed in the form of a scar, which was not apparent when the patient smiled because it was concealed in the upper lip . Patient satisfaction was recorded after 1-week of the procedure on a scale of 10 with score 1 for extremely unsatisfactory to 10 for a highly satisfactory score . Patient was highly satisfied with the treatment carried out and rated a score of 9 on a scale of 10 . The gingival display measured after 1-week was <1 mm with only interdental papilla being seen after forced smiling giving an esthetically pleasing appearance . Revaluation was further carried out after 1-month to see the stability of the results obtained . The gingival display noted after 1-month was 1 mm while smiling [figure 9]. After a period of 1-month he did not find any tension or pain while smiling [figure 10]. 1 mm of gingival display on smiling postoperatively after 1-month the postoperative picture after 1-month lip repositioning is a surgical way to correct gummy smile by limiting the retraction of the elevator smile muscles (e.g., zygomaticus minor, levator anguli oris, orbicularis oris, and levator labi superioris) and yet preserve the labial frenum . Local anesthesia (xylocaine 2% with epinephrine 1:80,000) was administered at the vestibular mucosa and lip from the maxillary right to left first molar . With the help of a sharp probe, bleeding points were induced at the mucogingival junction, which guided the first incision to be carried out . A partial thickness incision was made at the mucogingival junction from the mesial line angle of the right central incisor to the mesial line angle of the right first molar [figure 3]. A second partial thickness incision that ran parallel to the first incision and 1012 mm apical of the mucogingival junction the incisions were connected at the central incisor region without involving the maxillary labial frenum and at the right first molar region creating a quadrilateral outline [figure 4]. The epithelium was then carefully dissected within this outline [figure 5], leaving the underlying connective tissue exposed [figure 6]. The same procedure was carried out on the left side at the mucogingival junction by making an incision from mesial outline of left central incisor to the mesial outline of left first molar . The next partial thickness incision was the one parallel to the first incision and 1012 mm apical to the mucogingival junction . The second strip of epithelium was then removed by careful dissection [figure 7]. The first partial thickness incision on mucogingival junction from mesial line angle of right central incisor to mesial line angle of right first molar the second partial thickness about 810 mm above the first incision and two vertical incisions to join at both the ends the strip of the epithelium which was removed the partial thickness dissection which was carried out the same procedure was done on the left side leaving the intact frenum the parallel incision lines were approximated with interrupted stabilization sutures (silk 4/0) [figure 8]. Patient was discharged with all postsurgical instructions and medications for 5 days which included analgesic (ketorolac trimethamine 10 mg twice daily for 3 days), antibiotic (amoxicillin 500 mg tds for 5 days), along with cold packs extra orally to decrease postsurgical swelling . The 4 - 0 mersilk sutures were placed patient was recalled after 1-week for a follow - up . The patient reported with mild pain and tension at the surgical site during the 1 week after surgery . It was seen later that the suture area healed in the form of a scar, which was not apparent when the patient smiled because it was concealed in the upper lip . Patient satisfaction was recorded after 1-week of the procedure on a scale of 10 with score 1 for extremely unsatisfactory to 10 for a highly satisfactory score . Patient was highly satisfied with the treatment carried out and rated a score of 9 on a scale of 10 . The gingival display measured after 1-week was <1 mm with only interdental papilla being seen after forced smiling giving an esthetically pleasing appearance . Revaluation was further carried out after 1-month to see the stability of the results obtained . The gingival display noted after 1-month was 1 mm while smiling [figure 9]. After a period of 1-month he did not find any tension or pain while smiling [figure 10]. 1 mm of gingival display on smiling postoperatively after 1-month the postoperative picture after 1-month this case report describes an innovative surgical procedure to treat the excessive gingival display during smiling . The procedure is different from the conventional lip repositioning surgery which was first reported in the medical literature in 1973 by rubinstein and kostianovsky . Litton and fournier described gummy smile correction with lip repositioning surgery, including elevator muscle detachment in cases with a short upper lip . Miskinyar, in 1983, found no relapses for the 27 patients treated with myectomy and partial resection of either one or both of the levator labii superioris muscles bilaterally in lip repositioning surgery . In 2010, ishida et al . Reported a significant reduction in gingival exposure in 14 patients treated with levator labii superioris myotomy, subperiosteal dissection, and frenectomy . The main objective for preserving the maxillary labial frenum is that it prevents the midline being shifted thus guiding for an esthetically pleasing smile and also avoids the morbidity associated with the removal of maxillary labial frenum . The surgical procedure yielded great results by achieving a greater degree of gingival coverage to hide the gummy smile . The chief complaint of a gummy smile was completely resolved by modified lip repositioning surgical procedure . The quality of life was improved as the procedure met the esthetic demands of the patient . The demerits of the surgical procedure would include: (a) chance of recurrence of a gummy smile (b) decrease in the vestibular depth following surgery . Thus considering all the perspectives,
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Cxcl16, a newly identified chemokine, has been described in both transmembrane and soluble forms . Transmembrane cxcl16 (tm - cxcl16) is expressed on the surface of macrophages, dendritic cells, and monocytes where it functions as an adhesion molecule for cxcr6-positive immune cells [1, 3]. Tm - cxcl16 undergoes cleavage by the disintegrin - like metalloproteinases adam10 and adam17 before it is released in soluble form to the outside of the cell [4, 5]. Soluble cxcl16 (scxcl16) prompts migration of leukocytes expressing cxcr6 in a dose - dependent manner . Because cancer cell migration and metastasis share patterns with leukocyte trafficking, attention has been focused on the role of cxcl16 in cancer progression . Cxcl16 is expressed in various cancers, including pancreatic, prostate, breast, colorectal, and nonsmall cell lung cancer [8, 9]. More importantly, lu et al . Revealed a positive correlation between cxcl16 mrna expression and prostate cancer aggressiveness such that metastatic lesions expressed higher levels of cxcl16 mrna than the primary prostate tissues . Have shown that scxcl16 induces migration and proliferation of cxcr6-expressing prostate cancer cells [9, 10]. Moreover, matsushita et al . Reported that high preoperative serous levels of scxcl16 were associated with liver recurrence and poor prognosis in patients with colorectal cancer . Immunohistochemical staining data from patients with colorectal or renal cancer correlated better long - term prognosis with stronger cxcl16 staining in cancer tissues [12, 13]. These limited reports imply different functions for cxcl16 depending on the location of its expression in cancer patients . Breast cancer is the most common malignancy and the second leading cause of cancer - related death in american women . Despite survival rates having improved steadily since 1990 therefore, it is important to get a better understanding of the molecular mechanisms underlying breast cancer metastasis and to develop prognostic and therapeutic strategies . In this study, we explore the expression and function of cxcl16 in breast cancer cell lines that differ in aggressiveness . The breast cancer cell lines sk - br-3, mcf-7, and mda - mb-231 were obtained from american type culture collection (atcc) (rockville, md). The noncancerous human mammary epithelial cell line mcf-10a was purchased from bioleaf biotech (shanghai, china). All cell lines were cultured at 37c in dulbecco's modified eagle medium (hyclone, waltham, ma) supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 ug / ml streptomycin in a humid incubator with 5% co2 . Total rna was extracted by biozol reagent (bioflux, tokyo, japan) according to the manufacturer's instructions . Less than 2 ug rna was reverse - transcripted into cdna using reverse transcriptase (promega, beijing, china) and oligo(dt)18 (takara, dalian, china). Primers for cxcl16 were as follow: sense 5-ggcccaccagaagcatttac-3 and antisense 5-ctgaagatgccccctctgag-3. Primers for glyceraldehyde 3-phosphate dehydrogenase were as follows: sense 5-gaaggtgaaggtcggagtc-3and antisense 5-gaagatggtgatgggatttc-3. Pcr was performed with an iq4 multicolor real - time pcr detection system (bio rad, hercules, ca) using sso fast evagreen supermix (bio rad). Pcr protocol was performed as follows: denaturing for three seconds at 98c followed by forty amplification cycles of annealing and extension at 55c for fifteen seconds . Normalized lysates (30 ug) were separated by electrophoresis in 12% sds - page and electrotransferred onto polyvinylidene fluoride membrane (pvdf membrane, millipore, billerica, ma). The membrane was blocked with 5% nonfat milk in tris - buffered saline - tween (tbst, ph 7.6) at room temperate for 1 h and incubated overnight at 4c with cxcl16 antibody (abcam, cambridge, uk). After three washes with tbst, the membrane was incubated with horseradish peroxidase- (hrp-) conjugated igg . Cells were trypsinized and 10 cells were incubated with pe - conjugated cxcl16 antibody (r&d systems, minneapolis, mn) in a dark room for 45 min . After two washings with phosphate buffered solution (pbs), expression of transmembrane cxcl16 in cells was analyzed with a becton dickinson facscan using a software facs express 3 (de novo software, los angeles, ca). Cells were seeded with a volume of 200 ul (2,000 cells / well) into 96-well plates (corning). Every 24 h, mtt was added to the well with a final concentration of 0.5 mg / ml and subsequently incubated for 4 h at 37c . Supernate was discarded and 150 ul / well dmso was added . The optical densities (od) were measured at 490 nm with a microplate reader (bio rad). Migration and invasion assays were performed using a transwell chamber (8 um pore size, millipore) according to the manufacturer's instructions . Cell culture inserts for the invasion assay were precoated with matrigel (bd biosciences, bedford, ma) for 4 h at 37c . Cells were seeded into the upper chamber, while 1 ml complete medium was added into the lower chamber as a chemotaxin . After culture for 24 h, noninvading cells were removed with a cotton bud . Cells that migrated to the lower surface were fixed in 4% paraformaldehyde for 20 min and underwent giemsa staining . Five random fields were selected for cell counting under a light microscope (100; nikon, tokyo, japan). Caspase-3 was measured using the caspase-3 activity kit (beyotime, nanjing, china). Appropriate cells were incubated with 30 ul lysis buffer on ice for 30 min and then subjected to centrifugation at 13,000 rpm for 5 min . 10 ul of supernatant was incubated with ac - devd - pna at 37c for 5 h. the od values were detected at 405 nm . Cells were collected with edta trypsin and washed twice with phosphate buffer solution (pbs). In a 500 ul binding buffer, cells were stained with 5 ul annexin v and 5 ul 7 aad successively in the dark room . Apoptosis index is calculated by the percentage of cells with positive for annexin v and negative for 7-aad . The day before transfection, mda - mb-231 cells were seeded into a six - well plate at a density of 10 cells / well . After transfection in serum - free medium for 12 h, cells were further cultured in complete medium . Both cxcl16 and control plasmids were obtained from genepharma company (shanghai, china). Five - week - old female nod / scid mice were purchased from beijing hfk bioscience co., ltd . Cells were trypsinized into single cell suspension, and 3 10 cells were injected subcutaneously into the right - lower flank of each mouse . Tumor volumes were approximated using the formula v = [(/6) l w], where l was the longest axis and w was the shortest axis . All mice were sacrificed by cervical dislocation, and the tumors were removed and weighed . All data are expressed as the mean standard deviation (sd) and compiled using spss 16.0 (spss, chicago, il). One - way analysis of variance was used to determine statistical and significant differences between control and treated groups . Expression of cxcl16 was evaluated in mammary epithelial cell mcf-10a and in three breast cancer cell lines: sk - br-3, mcf-7, and mda - mb-231 . As shown in figure 1(a), cxcl16 mrna was highest in mcf-7 (3.038 0.436-fold), followed by sk - br-3 (1 0.548-fold), while the lowest expression was observed in mda - mb-231 (0.07 0.049-fold). Flow cytometry demonstrated that transmembrane cxcl16 was highest in mcf-7 and lowest in mda - mb-231 (figure 1(b)). Mcf-7 and sk - br-3 (with 32.25 2.63 and 59.75 6.94 migrated cells) showed less migration than mda - mb-231 cells (146.5 5.74, p <0.01) (figure 1(c)). Proliferation was the strongest in mda - mb-231 cells, while sk - br-3 cells and mcf-7 cells possessed less, though equal, proliferative capability (figure 1(e)). To further confirm the effect of cxcl16 on invasion, migration, and proliferation in these breast cancer cell lines elevated cxcl16 levels were confirmed by western blot or flow cytometry (figure 2(a)). Consistently, cxcl16-overexpressing cells showed significantly decreased cell migration (from 118 11.53 to 61.66 7.37, figure 2(b)) and invasion (from 84.0 5 to 48.66 4.04, figure 2(c)) compared with the control mock - transfected cells . However, mtt assay for cell proliferation revealed no difference between cells transfected with cxcl16 plasmids compared with the control mock - transfected cells (figure 2(d)). In addition, apoptotic index was increased from 3.5 2.03% to 5.9 2.87%, and the activity of caspase-3, a crucial enzyme in the apoptotic cascade, whose od value was enhanced from 0.65 0.032 to 0.89 0.121, indicated that increased cxcl16 expression facilitated caspase-3-dependent apoptosis (figure 2(e)). Meanwhile, to determine whether cxcl16 gene silencing would affect breast cancer progression, mcf-7 cells highly expressive for cxcl16 underwent stable knockdown of cxcl16 using shcxcl16, scrambled shrna (shnc), or liposomes only (mock). The efficiency of cxcl16 knockdown was confirmed by qrt - pcr, western blot, or flow cytometry which showed decreased cxcl16 expression in shcxcl16-transfected mcf-7 cells (figure 3(a)). Stable downregulation of cxcl16 expression in mcf-7 cells resulted in a significant increase in migration and invasion (figures 3(b) and 3(c)), but not proliferation (figure 3(d)). As shown in figure 4(a), cxcl16-overexpressing mda - mb-231 showed delayed tumor progression as compared to mda - mb-231 or the positive control (mcf-7) (p <0.05). Tumor volumes and weights for cxcl16-overexpressing groups were accordingly found to be significantly reduced (p <0.05) (figure 4(b)). Cxcl16 is a novel chemokine first cloned by maltoulin in 2000 . Like other chemokines, existing both in soluble form and as a transmembrane form, cxcl16 possessed functions seemingly more nuanced than other well - described chemokines . In soluble form it induces immunocyte chemotaxis, while in transmembrane form cxcl16 mediates cell - cell adhesion [1, 3, 4]. Previous findings from in vitro studies using exogenous scxcl16 suggested that scxcl16 promotes cell proliferation and invasion . Although matsushita et al . Associated high preoperative levels of scxcl16 with liver recurrence and poor prognosis in colorectal cancer patients, little was known about the functions of tm - cxcl16 except that high expression of cxcl16 in cancer tissues correlated with favorable prognosis in renal and colorectal cancer patients [12, 13]. These observed differences between tm - cxcl16 and scxcl16 indicate a complicated function for cxcl16 in cancer . We therefore sought to identify a specific function for tm - cxcl16 through in vivo and in vitro breast cancer models . In this study, we show for the first time that upregulation of cxcl16 suppresses migration and invasiveness of breast cancer cells in vitro and delays progression of tumor growth in vivo . These results reveal a protective function of cxcl16 in breast carcinogenesis and present valuable clues to better understanding of the mechanisms of breast cancer progression . Our measurements of both protein and mrna levels of cxcl16 reveal high expression in mcf-7 cell line, which are positive for estrogen receptor (er) and progesterone receptor (pr) and therefore considered less aggressive; levels were lower in the receptor triple - negative cancer cell line of mda - mb-231 which is considered more highly aggressive . This inverse correlation between cxcl16 expression and migration or invasion suggested that cxcl16 play another role as tumor suppressor in inhibiting the migration and invasiveness of breast cancer cells . As discussed above, tm - cxcl16 plays a pivotal role as a tumor suppressor on the development of breast cancer cells; that is, the high expression of cxcl16 or overexpression of cxcl16 significantly suppressed the invasion and migration and induced apoptosis of breast cancer cells; in contrast, low expression of cxcl16 or downregulation of cxcl16 promoted the invasion and migration of breast cancer cells . Supportively, the studies have reported that the reduced cell - cell adhesiveness is considered one morphological hallmark of malignant tumors, and homotypic adhesion was described to reduce the invasive potential of tumor cells . Consistent with previous reports, we found that cxcl16 and cxcr6 were coexpressed in breast cancer cells but to inverse extent . When expressed as transmembrane protein, cxcl16 could directly promote cell - cell adhesion by combining with cxcr6 on cell surface . Thus, we speculate here that transmembrane cxcl16 facilitates the cell - cell adhesion like other well described cell adhesion molecules, such as e - cadherin, which subsequently prevent the detachment of individual tumor cells from the tumor aggregation and ultimately inhibit cell migration and invasion . Heparin - like glycosaminoglycans are long unbranched polysaccharides consisting of a repeating disaccharide unit that expressed on proteoglycan components of cell surface and extracellular matrix . Cell surface glycosaminoglycans could bind positively charged regions on the protein and lead to enhanced local concentrations of chemokines . For example, heparin binds to the strong positive potential on cxcl12 and stabilizes the cxcl12 dimer on cell surface . Moreover, the presence of cell surface gags could enhance the activity of chemokines including mip-1a, rantes, or mip-1b by sequestrating them onto the cell surface . There also has been evidence that gag is important for cxcl16 recognition and that cxcl16 was able to react with heparin microarrays containing oligosaccharides . However, it remains unknown what the effect of gag - cxcl16 interactions is and how heparin - like glycosaminoglycan affects the activity of cxcl16 . Another significant finding was that overexpression of cxc16 enhanced apoptosis in mda - mb-231 in conjunction with upregulation of caspase-3, a crucial enzyme in endogenous apoptosis, suggesting cxcl16 promotes caspase-3-dependent apoptosis . Aberrant balance between proliferation and apoptosis is clearly implicated as one major characteristic in tumor development . However, in this study the apoptosis index or caspase-3 activity in mcf-7 cells is not determined, because the mcf-7 cell line was considered not to express the enzyme caspase-3 . We discovered no role for cxcl16 in the proliferation of breast cancer cell lines in our study, unlike other tumor suppressor studies . It has been speculated that tumor cell lines are normally transformed and have multiple genetic alterations, which may contribute to the noneffect of cxcl16 on the breast cancer proliferation . Egfr overexpression is well - studied in breast cancer, and egfr - driven signaling pathways such as pi3k / akt / mtor, jak / stat, and ras / raf / mapk were associated with cell proliferation and survival . Since egfr overexpression is common in breast cancer, the impact of cxcl16 alteration on proliferation may be masked by egfr overexpression or other genetic alterations . These are speculative explanations for how cxcl16 appears not to affect proliferation of breast cancer cells . In addition, since cxcl16 exists both as a transmembrane form and a soluble form, and the former could be cleavaged off into the soluble form, it is difficult to distinguish precisely which form is being studied in which cancer experiments . In fact, it has been reported that a molecule adam10 mediates the conversion of transmembrane cxcl16 into soluble cxcl16 dynamically [4, 5]. Therefore, in this study we defined cxcl16 as cellular cxcl16, which exists predominantly in the cell membrane . To characterize it, we employed flow cytometry to quantify cxcl16 localized on cell surfaces in living cancer cells . We report high cell surface expression of cxcl16 on living cells, in proportion to total cxcl16 expression by cells . Our data suggest that the transmembrane cxcl16 is involved in breast cancer cells with important clinical significance . In summary, the current study demonstrated that cellular cxcl16 expression was negatively correlated with the migration and invasion of breast cancer cells.
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Cases were identified through the hospital - based active surveillance system of the hfrs maintained by the korean army . Cases occurring from january 1, 2002, to january 1, 2004, were enrolled prospectively . For early detection of hfrs, patients with hfrs may have sudden onset of fever; experience pain in the head, abdomen, and lower back; and report bloodshot eyes and blurry vision . The patient's face, chest, abdomen, and back often appear flushed and red, as if sunburned . A confirmed case of hfrs is defined as a positive result on the high - density particle agglutination test . For each case, one control was selected from among the other patients at each hospital where the case - patient had been hospitalized . The control was matched with the case - patient according to unit, age at the time of hospitalization (3 years), date of hospitalization (3 months), and date of transfer to the present unit (3 months). If no suitable control could be found, the intervals around the case - patient's unit, age, date of hospitalization and date of transference were progressively widened until one or more potential controls were found . For each case - patient, all eligible controls were listed, and one suitably matched control was identified at random . As with the case - patients, the final decisions about each control patient's eligibility for the study were made on the basis of a detailed review of hospital records . Decisions about the eligibility of potential controls were made without knowledge of their vaccination status . Vaccine had to be received at least 3 weeks before hospitalization because of the time required for antibodies to develop and because the incubation period is 3 weeks on average (5). Estimates of the relative odds of hfrs associated with vaccination were estimated by using methods developed by mantel and haenszel (6), which are appropriate for matched designs . The protective effectiveness of the vaccine was estimated as 1 minus the relative odds associated with vaccine use, times 100 . Ninety - five percent confidence limits for the effectiveness were derived from the 95% confidence interval (ci) of the relative odds . Chicago, il). From january 1, 2002, to january 1, 2004, a total of 57 hfrs cases were identified among troops of the korean army . Of one of three deaths occurred in previously healthy, vaccinated (first and second doses) personnel . Twelve, 9, and 2 cases occurred in personnel who were vaccinated with one, two, and three doses, respectively . Most cases occurred in october (15.7%), november (35.7%), and december (17.1%), although disease also occurred during the spring and the summer (figure 2). Hemorrhagic fever with renal syndrome cases among republic of korea military personnel, by month of onset, january 2002 to january 2004 . Of the 54 persons identified with hfrs from january 2002 to january 2004, those who were vaccinated within 3 weeks of hospitalization were excluded from analysis . Because the effectiveness was calculated by comparing each dose (exactly one to three) with no vaccination, case - patients or controls not applicable to that comparison were excluded from each matched set . Finally, 41, 38, and 31 matched sets were formed for one, two, and three doses of the hantavirus vaccine, respectively (table). Estimates of vaccine effectiveness according to the number of doses received rose from 25% (95% ci 78% to 68%) for one dose to 46% (95% ci 35% to 78%) for two doses to 75% (95% ci 18% to 95%) for three doses . When recipients for whom 1 year had passed since their second dose were excluded, effectiveness of two doses increased markedly to 70% (95% ci 9% to 92%). The protective effectiveness of the vaccine strongly depends on the number of doses . In particular, effectiveness increased when persons for whom> 1 year had passed since their second dose were excluded, which suggests that the protective effect of the second primary vaccination does not persist beyond the period recommended for having the booster dose . In addition, we do not know whether the recommended immunization schedule was optimal for military personnel and farmers, groups for whom hantavirus vaccination is recommended . The vaccination schedule should be epidemiologically relevant, immunologically effective, operationally feasible, and socially acceptable (7). In a field study from the former republic of yugoslavia conducted by korean researchers, including the developers of the hantavirus vaccine (8), no case of hfrs was observed among 1,900 vaccinees, while 20 confirmed cases were observed among 2,000 nonvaccinated controls . Considering that our study showed low protective effectiveness for one or two doses, that no case of hfrs occurred in yugoslavian vaccinees before they received the full three doses was surprising . Because the case - control studies were not experimental, they may be subject to biases . The most important potential biases that might affect this kind of study are detection and selection bias . If all cases of hfrs were identified, no detection bias would occur . Because patients were identified prospectively by active surveillance, we believe that virtually all cases of hfrs diagnosed during the study period were identified . Selection bias may occur when controls do not represent the general population . In this study, controls were selected randomly from a list of potentially eligible controls by using a systemic algorithm . Confounding influences affect the results of a case - control study if controls differ from case - patients in characteristics related to risk of contracting the disease and likelihood of receiving the vaccine . Since the population of this study consisted of military personnel, bias due to sociodemographic differences may be negligible . Therefore, other candidate confounding factors determined by considering the military milieu were used as matching variables . Therefore, while point estimates show effectiveness, this finding could be due to chance . Of course, the range of point estimates in studies with relatively small samples can be wide, and wide confidence intervals that include zero are not uncommon in many studies on vaccine effectiveness (9). However, caution is appropriate in interpreting our estimates of vaccine effectiveness . Finally, this study represents a short - term (7.3 months average) evaluation of protective effectiveness of three doses of the hantavirus vaccine . To assess the long - term effectiveness, protection
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The activation of white phosphorus (p4) with transition metal (tm) complexes with the objective of generating organophosphorus compounds has been an ongoing research topic.1 for this purpose, an understanding of the p4 transformation pathway in the coordination sphere of transition metals is necessary . Thus, a variety of pn ligand moieties were stabilized to give insight into the stepwise p4 degradation and aggregation processes using wellestablished ligand systems such as the cp family (cp = cyclopentadienyl).1 however, over the last years, diketiminato (nacnac = l) ligand systems have gained increasing attention in mild p4 activations using m precursors: the initial p4 fixation step of an intact p4 tetrahedron at a metal center was achieved at an electronrich cu nacnac compound.2 in reactions with transition metal complexes of groups 53 and 810,4 products with modified [p2], [p4], [p4] and [p8] ligands, respectively, were obtained . So far, for nacnac systems, a [p3] ligand was found solely in compound [(lvr)2(cyclop3)] (r = n(tolyl)2, n=0,1; r = o(dipp), n=0) and [{lv(n(tolyl)2)}2(2:,cyclop3)] (a, scheme 1).3c selected examples of pntm complexes containing ligands l l (l = ch[chn(2,6ipr2c6h3)]2, l = ch[c(me)n(2,6me2c6h3)]2, l = ch[chn(2,6me2c6h3)]2, l = ch[c(me)n(2,6ipr2c6h3)]2, l = ch[c(me)n(2,6et2c6h3)]2). Bottom: comparison of the nacnac ligands used in this study, l, l and l.5 however, cyclop3 complexes of the type [lm(2:,p3)ml] have been structurally characterized using neutral, tridentate triphos (1,1,1tris(diphenylphosphinomethyl)ethane) and etriphos (1,1,1tris(diethylphosphinomethyl)ethane) ligands in different combinations with 3d metals (fe, co, ni) and 4d metals (rh, pd).6 the influence of the ligand substituents in femediated p4 transformation has recently been illustrated by a comparative study using a set of ligands ll (scheme 1).7 despite the application of the same reaction conditions, different products were obtained, which are sensitively dependent on small changes of the ligand substituents . 2,6diisopropylphenyl (dipp) substituents as the ligands aromatic flanking groups support the formation of the dinuclear complexes [(lfe)2(2:,p4)] (b)7 and [(lfe)2(2:,p2)2] (c).4c the latter was synthesized by the driess group.4c the ligands l and l only differ in their backbone substituents . However, for sterically less demanding 2,6dimethylphenyl (dmp) substituents, the formation of the tetranuclear complexes [(lfe)4(4:,,,p8)] [l = l (d1), l (d2)] with dimerized p4 units was observed.7 these results demonstrate that the product formation is affected by both the aromatic flanking groups and the ligand backbone substituents . Simultaneously, we investigated the [lco]mediated transformations of white phosphorus, which resulted in novel p4 and p3containing complexes (vide infra). In the meantime, driess and coworkers reported p4 activation by [lco] fragments leading to the neutral complexes [(lco)2(2:,p4)] [l = l (e1), l (e2)] (scheme 1).4b oneelectron reduction led to the monoanionic products [k(dme)4][(lco)2(2:,p4)] [l = l (f1), l (f2)] and transformed the [p4] middle deck into a [p4] ligand . Recently, wolf and coworkers have reported on [(bian)co]mediated p4 activations with a nacnacrelated bidentate redox noninnocent bian (1,2bis(2,6diisopropylphenylimino)acenaphthene) ligand system yielding compounds containing [p4] moieties.8 motivated by our first results with [lco] compounds, we speculated that the p4 activation outcome should be sensitive to the oxidation state of the precursor (co versus co). Additionally, we wanted to address the question of the ligands influence (l l) in comediated p4 activations and we were intrigued by the observed patom extrusion from the initially obtained p4 middle deck to form p3 compounds ., we report on the p4 activation by a formal co precursor yielding the monoanionic [k(thf)6][(lco)2(2:,p4)] (2) and [k(thf)6][(lco)2(2:,p3)] (3). Through a comediated p4 transformation at room temperature or under thermolytic conditions, the corresponding neutral relatives are obtained, which generate 2 and 3 selectively after subsequent oneelectron reduction . The redox chemistry of the products was investigated by cyclic voltammetry (cv), and their magnetic behavior was examined both in solution (evans method) and in the solid state (squid). The formal co precursor [k2(lco)2(2:,n2)] (1) was synthesized by a onepot reaction and was isolated as two different solvomorphs, 1solv (solv = nhexane9 or oet2).10 the xray structures of 1solv consist of two [lco] fragments bridged by a n2 unit . Two potassium atoms cover the n2 moiety and are coordinated in the phenyl pockets of the dipp substituents.11 the nn distance in 1nhexane / oet2 is 1.215(3) and 1.220(4), respectively, which is in line with the that (1.220(2)) of the previously reported [k2(lco)2(2:,n2)] [l = ch[c(tbu)n(2,6ipr2c6h3)]2].12 the presence of a [n2] moiety in 1nhexane is supported by raman spectroscopy (nn=1568 cm).9 the reaction of 1 with p4 proceeds by n2 evolution, showing that the formal [n2] species is reoxidized and revealing 1 as a formal dicobalt(0) starting material . Conducting the reaction in 1:1 stoichiometry leads to the complete consumption of p4 and the formation of a mixture of the monoanionic complexes [k(thf)6][(lco)2(2:,p4)] (2) and [k(thf)6][(lco)2(2:,p3)] (3), which were detected by h nmr spectroscopy.13 the appearance of a cyclop3 moiety in product 3 indicates that an extrusion of one p atom from the cyclop4 moiety in 2 is possible . However, if the reaction is conducted with two equivalents of p4, compound 2 is the only product found in the h nmr spectrum . The solid state structure of 22 thf reveals a salt consisting of two [k(thf)6] cations and two crystallographically distinguishable [(lco)2(2:,p4)] monoanions (figure 1).9 each anion is a centrosymmetric dicobalt complex that consists of two [lco] fragments bridged by a planar cyclop4 ligand . The pp distances amount to 2.1913(10)2.1951(10) in anion 1 and 2.1897(10)2.2004(10) in anion 2, respectively . These values correspond well with the cyclo[p4] moiety (2.178(1) and 2.207(1)) of the reported compound [(lfe)2(2:,p4)] (b).7 the central p4 ligands in 2 are almost square planar with interior angles of 86.07(3) and 93.92(3) in anion 1 and 86.38(3) and 93.62(3) in anion 2 . The cop distances range from 2.3362(7)2.4149(7) in anion 1 and 2.3441(7)2.4190(7) in anion 2 . Minor deviations within the atomic parameters of compound 22 thf and the related compounds [k(dme)4][(lco)2(2:,p4)] [l = l (f1), l (f2)] can be explained by small changes in the organic environment of the counter ion and the nacnac ligands of the complex monoanions . They may affect the coco distances and their coordination geometry (torsion angle between the coco axis and the plane formed by the nitrogen atoms and the methine carbon in the ligand backbone; figure 1 for graphical presentation of).14 anionic part of the molecular structure of 2 . Hydrogen atoms and aromatic flanking groups are omitted for clarity; thermal ellipsoids are drawn at 50% probability level . The torsion angle is depicted spanning between the coco axis and the plane formed by the nitrogen atoms and the methine carbon in the ligand backbone . Comparison of pp and coco atomic distances and torsion angles in anions of [k(solv)x][(lco)2(2:,p4)] (l = l (2)9, l (f1)4b, l (f2)4b). The monoanionic [(lco)2(2:,p3)] was obtained in two different solvomorphs [k(dme)4][(lco)2(2:,p3)]dme (3 a) and [k(thf)6][(lco)2(2:,p3)]2 thf (3 b). Both compounds are ionic and consist of solvent (dme or thf) molecules, one solventsaturated potassium counter ion, and the [(lco)2(2:,p3)] monoanion (figures 2 and 3). In both xray structures, the complex anions are built from two [lco] fragments bridged by a p3 triangle . In 3 a, the l ligand planes are almost parallel to each other with an dihedral angle of 2.00(7) (n1n2 versus n3n4). However, in 3 b, the ligand planes are in a twisted conformation with a dihedral angel of 74.2(4) (n1n2 versus n3n4, figure 2). Comparison of the anions in the molecular structures of 3 a (left) and 3 b (right). Hydrogen atoms and aromatic flanking groups are omitted for clarity; thermal ellipsoids of co and p atoms are drawn at 50% probability level; major component of disordered cyclop3 ligand is drawn in 3 a. anionic part of the molecular structure of 3 b. hydrogen atoms are omitted for clarity; thermal ellipsoids are drawn at 50% probability level . The different complex anion conformations may originate from packing effects directed by the unequally shaped counter cations . The cyclop3 middle deck is disordered over two positions in 3 a (occupancy 81:19).15 the middle deck in 3 b, however, is localized at one distinct position . As can be seen in table 2, the pp distances in 3 a are similar to the ones in the nacnac containing compound [{lv(n(tolyl)2)}2(2:,cyclop3)] (a),3c displaying a cyclo[p3] moiety . In 3 b, they compare better with the ones in [(triphos)co(2:,cyclop3)fe(etriphos)](pf6)2 (g).6b overall, they are in line with pp single bonds [for comparison: pp single bond in white phosphorus determined by xray diffraction: 2.209(5),16 electron diffraction: 2.1994(3),17 raman spectroscopy: 2.2228(5),18 and dft calculations: 2.1994(3) 17]. The cop distances in 3 a are between 2.2046(17) and 2.3684(8) and for 3 b in the range of 2.248(3) and 2.277(3). The coco distance in 3 a is 3.7359(5) and amounts to 3.724(2) in 3 b, which is slightly elongated compared to 2 (3.603 and 3.625, table 1). Comparison of selected atomic distances and angles in the [(lco)2(2:,p3)] anion in 3 a (major component) and 3 b, [{lv(n(tolyl)2)}2(2:,cyclop3)] (a)3c and the dication in [(triphos)co(2:,cyclop3)fe(etriphos)](pf6)2 (g).6b the h nmr spectra in [d8]thf display signals between 11.42 and 35.29 ppm for 2 9 and 8.15 and 12.85 ppm for 3, respectively . Except for the thf and dme signals, respectively, the h nmr spectra of 3 a, b do not deviate from each other . No signals are detected in the p{h} nmr spectra for 2 and 3 due to their paramagnetic nature . Their magnetic moment (eff) in [d8]thf solution (rt) was determined by the evans method: 3.90 b (2) and 3.51 b (3). In the solid state, these values are confirmed by squid measurements displaying a gradual decrease of the magnetic moment in the temperature range from 300 to 2 k of 3.80 to 3.30 b in 2 and 3.58 to 1.70 b in 3 a. therefore, the electronic structure of 2 is best described as containing a [p4] moiety bridging mixed valence co and co centers . This is in agreement with the previously reported compounds f1 and f2.4b compound 3, however, contains a [p3] ligand, which is bridged by two co metal centers . As mentioned above, starting from the formal co precursor 1, we obtained the compounds 2 and 3 as a mixture of products, the ratio of which is sensitively dependent on stoichiometry and reaction conditions . To discover an alternative approach, we targeted the use of the co starting material [lco(tol)] (4 a), which was speculated to yield the neutral analogues of 2 and 3 . Therefore, the co compound [lco(tol)] (4 a) was reacted with p4, and [(lco)2(2:,p4)] (5 a) was selectively formed . Metric parameters and the characterization of compound 5 a are discussed in detail below . Refluxing 5 a for three hours (110 c, toluene) gives rise to the loss of one phosphorus atom and the formation of [(lco)2(2:,p3)] (6),19 which was clearly characterized by mass spectrometry20 and h nmr spectroscopy.21 the dinuclear compound contains two [lco] fragments, and the bridging middle deck exhibits a savage disorder within its cyclop3 moiety . However, the initially localized electron density unambiguously displays triangleshaped cyclop3 constitution and enables an estimation of the pp distances in 6 (approx . These values are comparable with the ones found in 3 a (2.1674(13), 2.1790(16), 2.3303(17)) and 3 b (2.217(4), 2.224(4) and 2.237(4)) and are elongated compared to the ones in a (2.1658(10), 2.1804(9) and 2.2155(9)).3c the coco distance in 6 is 3.747 and therefore comparable to the ones in 3 a (3.7359(5)) and 3 b (3.724(2)), but elongated compared to its precursor complex 5 a (3.610, vide infra). The h nmr spectrum of 6 reveals signals between 20.06 and 12.68 ppm . The magnetic moment (eff) of 6 in c6d6 solution is 2.97 b at room temperature (evans method).22 this value is confirmed in the solid state by a squid measurement . A successive decrease from 2.7 to 2.0 b was measured in the temperature range from 300 to 2 k (see the supporting information). The electrochemical properties of the complexes 5 a and 6 were probed by cyclic voltammetry (cv) in thf solution containing bu4npf6 electrolyte (0.1 mol l, 295 k, see supporting information for further details).20 an irreversible oxidation was detected at e 1/2=0.34 v for 5 a and e 1/2=0.11 v for 6 (vs. cp2fe / cp2fe). The compounds 5 a and 6 each reveal one reversible reduction at e 1/2=1.62 v (vs. cp2fe / cp2fe). The complexes 2 9 and 3 confirm these values by the corresponding electrochemical behavior . For 3, an additional reduction event was monitored at 2.52 v (vs. cp2fe / cp2fe). We experimentally performed the reduction of 5 a and 6, respectively, with one equivalent of potassium graphite in thf at room temperature . The corresponding anionic compounds [k(thf)6][(lco)2(2:,p4)] (2) and [k(thf)6][(lco)2(2:,p3)] (3), respectively, are selectively and quantitatively formed, which was proven by h nmr spectroscopy of the crude reaction solution . On a preparative scale, the isolated yields obtained as single crystals are 41% for 2 and 62% for 3 . Consequently, regarding selectivity, this synthetic route is superior to the comediated p4 activation, which, in contrast, yielded a mixture of products . Three diketimines (lh, lh, lh) were synthesized to provide a comparable hybrid ligand set l l with backbone (r = h, me) and aromatic (ph*=dmp or dipp) substituents (scheme 1 and scheme 2), and to investigate the influence of the ligand design on the comediated p4 transformation . The [lco(tol)] [l = l (4 a), l (4 b), l (4 c)] starting materials were prepared in onepot reactions (see the supporting information). All conducted p4 activation reactions were performed under the same conditions ([lco(tol)]:p4=2:1, toluene, 23 h, rt) and yielded similar isolated products [(lco)2(2:,p4)] [l = l (5 a), l (5 b), l (5 c)]. The crystals of all the new compounds 5 a c were grown from saturated toluene solutions, and single crystal xray diffraction was performed . C are shown in figure s5 in the supporting information . As a representative, compound 5 a is presented in figure 4 a. its p4 moiety is rectangularly shaped, consequently spanned by two shorter and two longer pp atom distances . Together with two coordinating co atoms, the [p4co2] complex core builds a distorted octahedron . In 5 a c, the shorter pp atom distances are between 2.1256(6) and 2.1301(7) and the longer pp distances are between 2.2513(10) and 2.2980(7). Compared with a phosphorus single bond in the tetrahedral p4, the planar rectangularshaped p4 moieties in 5 a c contain a pair of shorter and a pair of elongated pp bonds . The coco distances in 5 a c are between 3.502 and 3.610 and, therefore, any bonding interaction can be ruled out . Due to the centrosymmetric molecular structure (p21/n in 5 a c), the ligands are parallel to each other . In 5 a c, the torsion angels (between the coco axis and the plane formed by the nitrogen atoms and the methine carbon in the ligand backbone) are between 3.40(6) and 12.32(6). In 5 b, c (and e1, 2 4b), the pp edges of the cyclop4 unit are nearly parallel or rectangular, respectively, compared to the nn axis of coordinating nitrogen atoms (compare (nnppshort)= 1 and (nnpplong)= 2, see figure 4 b). The structural parameters of 5 a c and e1, 2 are summarized in table 3 . A) molecular structure of the compound 5 a. hydrogen atoms and aromatic flanking groups are omitted for clarity; thermal ellipsoids are drawn at 50% probability level . The torsion angle is depicted, which spans between the coco axis and the plane formed by the nitrogen atoms and the methine carbon in the ligand backbone; b) view along co1co1 axis, revealing the angles 1 and 2, which span between the nn axis of coordinating nitrogen atoms and the edges of the cyclop4 unit . Comparison of pp and coco atomic distances in neutral [(lco)2(2:,p4)] [l = l (5 a), l (5 b), l (5 c), l (e1), l (e2)]. C display signals between 11.99 and 28.61 ppm and reveal their paramagnetic nature in solution . Therefore, no signals are detected in their p{h} nmr spectra . Their magnetic moment (eff) in solution (rt) was determined by the evans method: 3.02 b (5 a 22 in c6d6), 2.42 b (5 b in c6d6), 1.84 b (5 c in [d8]thf). In the solid state, however, the squid measurements of 5 a and 5 b display diamagnetic behavior in the temperature range of 2300 k. their electronic structure in the solid state is best described as two antiferromagnetically coupled co centers bridged by a [p4] ligand similar to the previously reported compounds e1, 2.4b in solution, exclusively one signal set for the ligand is observed in the h nmr spectrum of 5 a c, respectively, suggesting the integrity of each dinuclear compound in solution on the nmr time scale (figure s15). The electrochemical properties of the complexes 5 a and 6 were probed by cyclic voltammetry (cv) in thf solution containing bu4npf6 electrolyte (0.1 mol l, 295 k, see supporting information for further details).20 an irreversible oxidation was detected at e 1/2=0.34 v for 5 a and e 1/2=0.11 v for 6 (vs. cp2fe / cp2fe). The compounds 5 a and 6 each reveal one reversible reduction at e 1/2=1.62 v (vs. cp2fe / cp2fe). The complexes 2 9 and 3 confirm these values by the corresponding electrochemical behavior . For 3, an additional reduction event was monitored at 2.52 v (vs. cp2fe / cp2fe). We experimentally performed the reduction of 5 a and 6, respectively, with one equivalent of potassium graphite in thf at room temperature . The corresponding anionic compounds [k(thf)6][(lco)2(2:,p4)] (2) and [k(thf)6][(lco)2(2:,p3)] (3), respectively, are selectively and quantitatively formed, which was proven by h nmr spectroscopy of the crude reaction solution . On a preparative scale, the isolated yields obtained as single crystals are 41% for 2 and 62% for 3 . Consequently, regarding selectivity, this synthetic route is superior to the comediated p4 activation, which, in contrast, yielded a mixture of products . Three diketimines (lh, lh, lh) were synthesized to provide a comparable hybrid ligand set l l with backbone (r = h, me) and aromatic (ph*=dmp or dipp) substituents (scheme 1 and scheme 2), and to investigate the influence of the ligand design on the comediated p4 transformation . The [lco(tol)] [l = l (4 a), l (4 b), l (4 c)] starting materials were prepared in onepot reactions (see the supporting information). All conducted p4 activation reactions were performed under the same conditions ([lco(tol)]:p4=2:1, toluene, 23 h, rt) and yielded similar isolated products [(lco)2(2:,p4)] [l = l (5 a), l (5 b), l (5 c)]. The crystals of all the new compounds 5 a c were grown from saturated toluene solutions, and single crystal xray diffraction was performed . The molecular structures of 5 a c are shown in figure s5 in the supporting information . As a representative, compound 5 a is presented in figure 4 a. its p4 moiety is rectangularly shaped, consequently spanned by two shorter and two longer pp atom distances . Together with two coordinating co atoms, the [p4co2] complex core builds a distorted octahedron . In 5 a c, the shorter pp atom distances are between 2.1256(6) and 2.1301(7) and the longer pp distances are between 2.2513(10) and 2.2980(7). Compared with a phosphorus single bond in the tetrahedral p4, the planar rectangularshaped p4 moieties in 5 a c contain a pair of shorter and a pair of elongated pp bonds . The coco distances in 5 a c are between 3.502 and 3.610 and, therefore, any bonding interaction can be ruled out . Due to the centrosymmetric molecular structure (p21/n in 5 a c), the ligands are parallel to each other . In 5 a c, the torsion angels (between the coco axis and the plane formed by the nitrogen atoms and the methine carbon in the ligand backbone) are between 3.40(6) and 12.32(6). In 5 b, c (and e1, 2 4b), the pp edges of the cyclop4 unit are nearly parallel or rectangular, respectively, compared to the nn axis of coordinating nitrogen atoms (compare (nnppshort)= 1 and (nnpplong)= 2, see figure 4 b). The structural parameters of 5 a c and e1, 2 are summarized in table 3 . A) molecular structure of the compound 5 a. hydrogen atoms and aromatic flanking groups are omitted for clarity; thermal ellipsoids are drawn at 50% probability level . The torsion angle is depicted, which spans between the coco axis and the plane formed by the nitrogen atoms and the methine carbon in the ligand backbone; b) view along co1co1 axis, revealing the angles 1 and 2, which span between the nn axis of coordinating nitrogen atoms and the edges of the cyclop4 unit . Comparison of pp and coco atomic distances in neutral [(lco)2(2:,p4)] [l = l (5 a), l (5 b), l (5 c), l (e1), l (e2)]. The h nmr spectra of the compounds 5 a c display signals between 11.99 and 28.61 ppm and reveal their paramagnetic nature in solution . Their magnetic moment (eff) in solution (rt) was determined by the evans method: 3.02 b (5 a 22 in c6d6), 2.42 b (5 b in c6d6), 1.84 b (5 c in [d8]thf). In the solid state, however, the squid measurements of 5 a and 5 b display diamagnetic behavior in the temperature range of 2300 k. their electronic structure in the solid state is best described as two antiferromagnetically coupled co centers bridged by a [p4] ligand similar to the previously reported compounds e1, 2.4b in solution, exclusively one signal set for the ligand is observed in the h nmr spectrum of 5 a c, respectively, suggesting the integrity of each dinuclear compound in solution on the nmr time scale (figure s15). We reported different [lco]mediated p4 activations yielding neutral complexes [(lco)2(2:,p4)] (l = l, l, l), each containing a similar rectangularshaped [p4] moiety . In contrast to the p4 activation by lfe compounds, for the co system, the ligand substituents (l l) do not alter the reaction outcome . For the ligand system l, we demonstrate that one p atom can be extruded thermolytically to generate an unprecedented neutral cyclo[p3]containing compound [(lco)2(2:,p3)]. As a novel approach, we present the p4 transformation with a formal [lco] precursor, which generates corresponding monoanions with cyclo[p4] and cyclo[p3] ligands as a mixture of products . As a service to our authors and readers, this journal provides supporting information supplied by the authors . Such materials are peer reviewed and may be reorganized for online delivery, but are not copyedited or typeset . Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
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Phenylketonuria (pku) is an autosomal recessive inborn error of phenylalanine metabolism, which is caused by mutation in phenylalanine hydroxylase (pah) gene . Most of the pah mutations are missense mutations (67%), which are followed by small or large deletions (13%). We reported a patient with classic pku and his parents harboring a large deletion in exon 3 (ex3del4765) of pah gene . This finding may help improve early detection, differential diagnosis, genetic counseling, and even treatment of patients with pku . Phenylketonuria (pku) is caused due to various genetic alterations of phenylalanine hydroxylase (pah) enzyme, which is involved in conversion of l - phenylalanine (l - phe) to l - tyrosine (l - tyr) in phenylalanine metabolic pathway . The classic pah deficiency is considered, when the serum concentration of unchanged phenylalanine (phe) crosses the level of 1200-mol / l (1). The phe concentration in the range of 600 to 1200-mol / l is diagnosed as mild pku, and values less than 600-mol / l are classified as hyperphenylalaninemia (hpa).when the treatment program of a patient with pku did not start at the early weeks of neonatal period developmental delay, mental retardation and microcephaly are caused by accumulation of toxic byproducts of phe within its metabolic pathway . Pah gene is located on chromosome 12q23.2, spanning 171 kbp and includes 13 exons, which encode a polypeptide of 452 amino acids (2). More than 520 various pathogenic mutations have been reported in all 13 exons of pah gene which could be found in the pah mutation analysis consortium database . Missense mutations constitute 67% of the total pah mutations, which could result in different clinical manifestations based on their effects on structure and function of pah enzyme . Small or large exon deletions are the second frequent genetic alterations of pah gene, which comprises 13% of the total mutations listed in pah mutation database (http:// www.pahdb.mcgill.ca). Although complete deletion of exon 3 of pah gene has been previously reported in european patients, to our knowledge, it has not been identified among asian population, especially in iranian patients with pku yet . This report aimed to describe the same but the first iranian patient with classic pku who was homozygous for deletion of pah exon 3 . A 15-day - old male was referred to genomic research center in summer 2014 in tehran due to high level of blood phe detected in the routine national framework of pku neonatal screening (table 1). His parents were first cousins with no family history of pku (figure 1). Due to persistent high level of phenylalanine in blood (1404-mol / l), molecular genetic analysis was performed to confirm pku diagnosis in the child . After obtaining approval from the medical ethics committee of shahid beheshti university of medical sciences, and informed consent from patient family, peripheral blood samples from the patient and extracted genomic dna was amplified by polymerase chain reaction (pcr) technique by abi geneamp 9700 device, using pah gene primers amplifying exons 1 - 13 and exon - intron boundaries . Then, pcr products were sequenced using the abi prism 3130 genetic analyzer (applied biosystems, foster city, ca, usa) (primer sequences and pcr programs are available upon request). During pcr of pah exons in the patient, further analysis was conducted by designing two primers within the flanking introns of exon 3 (introns 2 and 3). In normal individuals, the segment size between two primers is 5885 bps (which does not amplify in regular pcr). In our patient, a 1120 bps segment was amplified (figure 2) that demonstrated a large deletion of 4765 bps (g.21560 - 26324del4765) containing exon 3, and results were confirmed by sanger sequencing (figure 3) too . As a result, pku is an autosomal recessive metabolic disorder caused by various molecular alterations in either pah gene or the gene encoding its cofactor, tetrahydrobiopterin (bh4). Multiple pathogenic and nonpathogenic variations have been reported in all 13 exons of pah gene . Deletions constitute 13% of all kinds of mutations identified in pah gene, however large exon deletions comprise less than 7% of all the deletions . We reported a new case of 4765 bps deletion (ex3del4765) encompassing the entire exon 3 of pah gene, which has been never described in asian and iranian patients . This deletion initially was identified in patients with pku from czech republic by performing mlpa (multiplex ligation - dependent probe amplification) and long range pcr . Also, it has been reported in patients with pku of other european populations such as italian, polish, and slovene, which could be an example of founder effect (3 - 5). Ex3del4765 is produced as a result of intra - chromosomal, unequal homologous recombination between alu - alu repeats, which is the major cause of large genomic rearrangements . Alu repeats are interspersed repetitive elements comprising 10% of the total human genome, and are found in untranslated regions, introns, and even interagenic regions of genome (6). Exon 3 of pah gene is involved in encoding the regulatory domain of pah enzyme (7), which lies at the n - terminal of the protein . Regulatory domain spans within the 1/3 of the total length of the n - terminal of pah protein and contains an auto - regulatory sequence (ars). Ars plays a fundamental role in the prevention of substrate from binding to enzyme active site (8). Ars also modulates the entrance of substrate, bh4, and catecholamine inhibitors to control the phe activation . Regulatory domain facilitates access of both substrate and cofactor through interaction with active site of catalytic domain, therefore either partial or complete deletion of it, could interfere with its proper function (9).considering the direct effects of various pathogenic mutations on the pah enzyme activity and the serum level of phe, accurate identification of the pah gene mutations could have a determining role in treatment schedule of pku patients . Despite the lack of enough investigations, it is demonstrated that some of the pah gene mutations are more responsive to bh4 therapy (10, 11). Moreover, determination of specific pah gene mutation in patients can lead to early initiation of phe - free regimen, which can prevent future severe neurological complications . Ascertaining the precise type of underlying molecular defect in pah gene, particularly those occurring in regulatory domain, not only confirms the diagnosis, but also could help health care providers to afford the best treatment program . In addition, identification of the mutation type could help family genetic counseling, especially in the prenatal diagnosis (pnd) of future pregnancies and this is one of the strong points of our study . Finally, discovering the deletion of exon 3 in pah gene in iranian population could assist us in better molecular diagnosis of patients with pku and their family members.
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Our clinical unit has recently offered mental health care at asylum seekers, rescued from ships from the horrors of wars, as have many other psychiatric facilities in sardinia, sicily and greece . In fact the number of migrants crossing the mediterranean towards europe has dramatically increased in 2015; as the number of incidents and deaths [2, 3]. This editorial summarizes our activity and the results of our work and highlights some critical aspects that hinder the care to asylum seekers with stress related disorders . Screening for mental distress was performed in all migrant joint three camps in sardinia over january september 2015 using k6, short screening scale for post traumatic stress disorder (ptsd)(french, english and arabic validated versions) and with an interview on wellness conducted by psychologists with cultural facilitators . People with psychiatric needs have been treated and evaluated at the start of treatment and three months later by means of cgi - s . In a sample of 860 asylum seekers (21.7% female, age: 36.9+/-14.7), we found that 190 (22.1% of sample, 22.6% female, age; 38.5+/-12.9) were positive for at least one screener; seventy - five (8.7%, 24% female, age 36.9+/-13.3) had a diagnosis of depressive or bipolar dsm-5 disorder (md) and 56 (7.6%, 25% female; age 39.4+/-12.6) of ptsd or ptsd plus md . Syrians showed the higher risk of ptsd against other groups (or=6.24, ic95% 1.20 - 28.0). Psychiatric disorders were treated with: antidepressants (64.1%) mood stabilizers (24.4%); antipsychotics (4.5%) and/or psychological support (100%). After three months of treatment: 51 treated people (26.8%) had left the camps without giving any explanation; some were stopped by illegally trying to escape from italy . Those who remained in the hosting facilities had psychopathological conditions worse than those that had gone away, cgi - s score 3.86+/-0.8 vs 3.45 + /-0.9 (df 1,188, 189, f= 9.15, p=0.003); 70.8% of those remaining declared that they should not be officially registered in italy as destination of arrival because under european union dublin regulation they have to stay in the country of first asylum; 53.1% had relatives in northern europe that they wanted to reach . In those who stay, cgi - s score change from 3,86+/-0.8 to 3.69+/-0.9 at three months (df 1,276, 272, f= 2, 77, p=0.010). Only 8 (8.3%) showed a significant clinical improvement, scoring 2 points or less at gci - s . Dissatisfaction and feeling of prisoners because they could not join relatives may have had a negative impact on outcome . Studies have shown that social support influenced long - term response to trauma and the lack of social contacts in exile predicted the maintenance of ptsd symptoms . Thus in severe ptsd, with the experience of torture and seeing family members killed, staying with surviving relatives in stable conditions would be an important part of treatment . From this point of view, the dublin regulation seems not to be in agreement with articles 23 and 25 of the un convention on the rights of persons with disabilities, which the european union has signed, because in these rules reunification with families is taken into consideration but it is impossible to do due to the long burocracy required (and in fact no migrant asked for reunification). This condition is a de facto denial of the right to appropriate treatment for asylum seekers with ptsd with doubt of illegality in face to the un convention.
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Papillary thyroid carcinoma (ptc) is the most frequent type of thyroid malignancy, and the usual metastasis sites include the locoregional lymph nodes . Distant metastasis of ptc is rare and usually involves the lungs, liver, bones and brain . The skin metastasis of ptc is a very rare condition, and the scalp is the most frequent cutaneous area which is involved . Other cutaneous areas that are involved with less frequency are the cheeks, shoulders, arms, abdomen and thighs . A 64 year - old female with past medical history of metastatic papillary thyroid carcinoma (ptc) with both liver and lung metastasis presented with a 2.5 3 cm erythematous tender nodule in right parietal scalp of two months duration . She had a past medical history of total thyroidectomy and also had received repeated doses of radioactive iodine (rai) therapy in the past two years . Her last dose of rai131 (200 mci) was three weeks prior to the time of presentation . She also had a history of subglottic mass as a result of thyroidal carcinoma involvement with severe tracheal stenosis, and had undergone tumoral resection and tracheal anastomosis and partial hemilaryngectomy two years ago because of subglottic involvement by thyroidal carcinoma . High resolution computed tomography (hrct) demonstrated multiple metastatic nodules in both lungs and loculated left side pleural effusion consistent with advanced metastatic involvement . A differential diagnosis of pillar cyst and metastasis was given by dermatologist for scalp lesion . Histopathologic examination of the lesion revealed skin tissue with tumoral involvement of the dermis composed of numerous follicles (likely thyroid follicles), lined by relatively large cuboidal epithelial cells with round nuclei, fine chromatin pattern, little granular cytoplasm and eosinophilic colloidal material within some of follicles, and mitosis, which was consistent with metastatic thyroidal carcinoma [figures 1 and 2]. She was discharged with suppressive therapy with levothyroxine and referred to oncology clinic for continuation of the treatment plan . Scalp lesion biopsy showing skin tissue with tumoral infiltration of dermis by numerous follicles (likely thyroid follicle), consistent with metastatic thyroidal carcinoma (h and e, 40) scalp metastasis of thyroidal carcinoma composed of numerous follicles lined by relatively large cuboidal epithelial cells with round nuclei, fine chromatin, little granular cytoplasm and eosinophilic colloid material within some of follicles with mitotic division (h and e, 400) the skin metastasis of ptc is mostly associated with aggressive and disseminated disease and shows a poor outcome . Some authors suggested that the average patient survival time after discovering of skin metastasis is 19 months . Here, we have reported a case of scalp metastasis of ptc in a patient with metastatic pulmonary and liver involvement . Metastasis of thyroid cancer should be considered in the differential diagnosis of scalp tumors in any patient with a history of thyroidal carcinoma; and, excisional skin biopsy is necessary for confirming the diagnosis.
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